XVI Congreso Chileno de HematologiacuteaVI Congreso de Medicina TransfusionalCoquimbo Septiembre 2008
Hemostasia y Trombosis iquestHacia un Modelo Integrador
Diego MezzanoDepto Hematologiacutea‐OncologiacuteaP Universidad Catoacutelica de Chile
MacFarlane RG Nature 1964MacFarlane RG Nature 1964ldquoldquoEnzyme CascadeEnzyme Cascaderdquordquo
Davie EW and Ratnoff OD Science 1964Davie EW and Ratnoff OD Science 1964ldquoldquoWaterfall SequenceWaterfall Sequencerdquordquo
Broze JG GJ
Osterud B and Rapaport SI PNAS 1977
Broze GJ Jr
Broze GJ Jr et alBiochemistry 1990
Broze GJ Jr
Broze GJ Jr
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
MacFarlane RG Nature 1964MacFarlane RG Nature 1964ldquoldquoEnzyme CascadeEnzyme Cascaderdquordquo
Davie EW and Ratnoff OD Science 1964Davie EW and Ratnoff OD Science 1964ldquoldquoWaterfall SequenceWaterfall Sequencerdquordquo
Broze JG GJ
Osterud B and Rapaport SI PNAS 1977
Broze GJ Jr
Broze GJ Jr et alBiochemistry 1990
Broze GJ Jr
Broze GJ Jr
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Osterud B and Rapaport SI PNAS 1977
Broze GJ Jr
Broze GJ Jr et alBiochemistry 1990
Broze GJ Jr
Broze GJ Jr
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Broze GJ Jr et alBiochemistry 1990
Broze GJ Jr
Broze GJ Jr
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Broze GJ Jr
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Gailani D Broze GJ Science 1991Naito K Fujikawa K JBC 1991
Broze GJ Jr
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Baglia FA Walsh PN Biochem 1998
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Insuficiencias del ModeloExperimentos in vitro usan fosfoliacutepidos que no replican resultados obtenidos con membranas celulares ricas en factores adicionales
Las plaquetas tienen una membrana rica en receptores y factores de la coagulacioacuten superficie oacuteptima para el ensamble de los complejos multimoleculares de la coagulacioacuten
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Copyright copy2002 American Society of Hematology Copyright restrictions may apply
Brummel K E et al Blood 2002100148-152
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Platelets are activated prior to the burst of thrombin generation
Roberts HR et al
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Platelets are required for thrombin generation in the model system
Monroe DM et al ATVB 2002
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Cell‐based Model of Thrombin GenerationCell‐based Model of Hemostasis
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Initiation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Amplification
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Propagation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Coagulation
A cell‐based model of thrombin generation Roberts HR et al Sem Thromb Haemost 2006
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
ABSTRACT Arterial thrombosis is considered to arisefrom the interaction of tissue factor (TF) in the vascular wallwith platelets and coagulation factors in circulating bloodAccording to this paradigm coagulation is initiated after avessel is damaged and blood is exposed to vessel‐wall TF Wehave examined thrombus formation on pig arterial media(which contains no stainable TF) and on collagen‐coated glassslides (which are devoid of TF) exposed to flowing nativehuman blood In both systems the thrombi that formed duringa 5‐min perfusion stained intensely for TF much of which wasnot associated with cells Antibodies against TF caused 70reduction in the amount of thrombus formed on the pigarterial media and also reduced thrombi on the collagencoatedglass slides TF deposited on the slides was active asthere was abundant fibrin in the thrombi Factor VIIai apotent inhibitor of TF essentially abolished fibrin productionand markedly reduced the mass of the thrombi Immunoelectronmicroscopy revealed TF‐positive membrane vesicles thatwe frequently observed in large clusters near the surface ofplatelets TF measured by factor Xa formation was extractedfrom whole blood and plasma of healthy subjects By usingimmunostaining TF‐containing neutrophils and monocyteswere identified in peripheral blood our data raise the possibilitythat leukocytes are the main source of blood TF Wesuggest that blood‐borne TF is inherently thrombogenic andmay be involved in thrombus propagation at the site ofvascular injury
Giesen PLA et al PNAS 1999
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Blood‐borne TF iquestde doacutende viene
Monocitos radic
Granulocitos
Endotelio
Micropartiacuteculas radic
Plaquetas radic‐
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Table 3 Plasma markers of oxidative stress endothelial activationdysfunction and haemostatic activation in patients with chronic renal failure and healthy controls (Kidney Int 2001 601844-50)
Plasma concentration of Patients (n = 64)
Controls(n = 16 ndash 40)
p=
TBARS (micromolL) 198 plusmn 048 155 plusmn 039 0009AOPP (mmol eq chloramine T) 281 (45-915) 121 (14-414) 00001von Willebrand factor () 182 plusmn 78 116 plusmn 46 00001Soluble thrombomodulin (ngmL) 157 plusmn 21 57 plusmn 045 00001Soluble ICAM-1 (ngmL) 301 (174-508) 233 (179-275) 00001TAT (microgL) 33 (094-142) 21 (084-47) 003PF1+2 (nmolL) 30 plusmn 11 18 plusmn 08 00001PAP (microgL) 874 (146-2302) 475 (321-805) 00001FnDP (ngFEmL) 675 (118-3622) 173 (9-543) 00001FgDP (ngFEmL) 391 (20-5875) 89 (20-236) 00001
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
0 0
0 2
0 4
0 6
0 8
1 0
1 2
1 2 3 4
TFA
CTI
N N
OR
MA
LIZE
D R
ATI
O(a
rbitr
ary
units
)
H E A L T H YIN D IV ID U A L S
T O T A LC R F
C R FP R E -D IA L YS IS
C R FD IA L YS IS
p lt 0 0 0 2 5
p lt 0 0 0 0 1
p lt 0 0 0 5
BASAL MONOCYTE TF EXPRESSION IS INCREASED IN CRF PATIENTSCOMPARED WITH HEALTHY INDIVIDUALSA
B
ACT (462 bp)
TF (282 bp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Platelet‐associated tissue factor contributes to the collagen‐triggered activation of blood coagulation Zillmann A Luther T Muumlller I Kotzsch M Spannagl M Kauke T Oelschlaumlgel U Zahler S Engelmann BBiochem Biophys Res Commun 2001 Feb 23281(2)603‐9
Platelet activation induces cell‐surface immunoreactive tissue factor expression which is modulated differently by antiplatelet drugs Camera M Frigerio M Toschi V Brambilla M Rossi F Cottell DC Maderna P Parolari A Bonzi R De Vincenti O Tremoli EArterioscler Thromb Vasc Biol 2003 Sep 123(9)1690‐6
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Escaping the Nuclear Confines Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets Cell Volume 122 Issue 3 Pages 379 - 391M Denis N Tolley M Bunting H Schwertz H Jiang S Lindemann C Yost F Rubner K Albertine K Swoboda
Este artiacuteculo dio sustento cientiacutefico a otros previos que reportaban siacutentesis de IL1β bcl‐2 y PAI‐1 por las plaquetas
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
11 Evidence of TF synthesis by human plateletsEvidence of TF synthesis by human platelets
2 Is TF present in non‐stimulated circulating platelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Classical RTClassical RT‐‐PCR reveals that human PCR reveals that human platelets platelets express TFexpress TF‐‐mRNA mRNA
200 bp
600 bp
1500 bp
LPS‐stimulated PBMC (2 h) increase dramatically TF‐mRNA expression but also contain GPIbα‐mRNA denoting contamination with platelets
β-actin
GPIbαβ-a
ctin
GPIbα
200 bp
600 bp
2000 bp
Stimulated (5mM TRAP 15 min) human platelets free of monocytes express TF-mRNA after activation and frequentlyhelliphellip even without stimulation
CD14CD14
TF
TF
Resting Stimulated
(J Exp Med 2006 2032433 Blood 2007 1095242)
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
350bp
50 bp
Plat β
‐actin
PBMC β
‐actin
PBMC T
F
Plat T
F
RTRT‐‐PCR TRAPPCR TRAP‐‐activated platelets (15 min) but not activated platelets (15 min) but not PBMC of one individual express TFPBMC of one individual express TF‐‐mRNAmRNA
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
604 kDa
472 kDa
351 kDa
TRAP ndash + ndash + Puromycin ndash ndash + +
Metabolic radioMetabolic radio‐‐labeling with labeling with 3535SS‐‐methionine methionine demonstrates TF neodemonstrates TF neo‐‐synthesis by platelets synthesis by platelets
TF synthesis (asymp47 kDa) by non stimulated platelets is enhanced with activation
(band of asymp60kDa) and inhibited by puromycin
IP polyclonal α‐TF
(Blood 2007 1095242)
TRAP ndash ndash + +Puromycin ndash + ndash +
620 kDa
475 kDa
325 kDa
IP MoAb α‐TF (Am Diag 4509)
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
1 Conclusions1 Conclusions
Isolated human platelets express TF-mRNA and neo-synthesize the protein These phenomena were observed in non-stimulated conditions but mainly after activation
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
1 Evidence of TF synthesis by human platelets
22 Is TF present in nonIs TF present in non‐‐stimulated circulating stimulated circulating
plateletsplatelets
3 TF‐dependent pro‐coagulant activity of human platelets
Possible mechanisms of TF activation
4 Is this TF hemostatically relevant
OUTLINEOUTLINE
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
60 kDa
47 kDa
30 KDa
hrTFN‐S 15 60min min
Western Blot of membrane fractions of nonWestern Blot of membrane fractions of non‐‐stimulated (Nstimulated (N‐‐S) and TRAPS) and TRAP‐‐activated plateletsactivated platelets
(Blood 2007 1095242)
Consistently TF protein was present in nonConsistently TF protein was present in non‐‐stimulated plateletsstimulated platelets
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Pearson Coeff 01855
TF GPIbα
Non‐stimulated non‐permeabilized human platelets express TF protein which appears centralized in relation to GPIbα
Pearson Coeff 03188
TRAP‐stimulation strikingly enhances the immuno‐reactivity of TF and its co‐localization with GPIbα
GPIbαTF
TF GPIbα
Merge
Merge
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
This apparent externalization of TF is also observed This apparent externalization of TF is also observed after membrane biotinilation and TF after membrane biotinilation and TF immunoprecipitationimmunoprecipitation
IP polyclonal‐αTF
83 kDa
642 kDa
WB Streptavidin‐HRP 47 kDa
37 kDaTF is transferred to plasma membrane after TRAP stimulation
WB MoAb α‐TF 4509 47 kDa
N‐S 30 60 90min min min
Again TF is present in non‐stimulated platelets
(Blood 2007 1095242)(J Thromb Haemost 20075(Suppl 2) O‐W‐074)
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
GPIbαTF VisIn contrast resting nonIn contrast resting non‐‐permeabilized PBMC do not exhibit permeabilized PBMC do not exhibit membrane TF Platelets always present in these membrane TF Platelets always present in these preparations contain TF copreparations contain TF co‐‐localized with GPIblocalized with GPIbαα
Merge
GPIbα
TF
TF
GPIbα
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
TFGPIbα
Merge
GPIbα TF
Merge
GPIbα TF
Merge
GPIbα TF
Merge
6 microM
However TF protein appears in LPS‐stimulated PBMC (2 h) Again high expression of TF‐GPIbα is observed in ldquocontaminatingrdquo platelets
Blood 2007 1095242
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
TFTF‐‐related prorelated pro‐‐coagulant activity (PCA) is not coagulant activity (PCA) is not inhibited by puromycin during a 2inhibited by puromycin during a 2‐‐h period denoting h period denoting that PCA depends on already stored TF in plateletsthat PCA depends on already stored TF in platelets
0 25 50 75 100 1250
10
20
30
Time (minutes)
Pro
coag
ulan
t act
ivity
(AU
2x
107 p
late
lets
)
Puromycin
Control
(Blood 2007 1095242)
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
2 Conclusions2 Conclusionsa TF is demonstrated in N-S platelets by western blotting
membrane biotinilation with immunoprecipitation and immunofluorescence-confocal microscopy
b In contrast surface exposure of TF in PBMC is detected only after 2-hour stimulation with LPS
c TF activity is not inhibited by simultaneous incubation with puromycin suggesting that TF is already stored in platelets
d These findings support the idea that human platelets contain stored TF likely synthesized by circulating platelets or alternatively derived from megakaryocytes
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Thrombus Formation In Vivo
Furie B and Furie B N Engl J Med 2008359938-949
Furie B and Furie B N Engl J Med 2008359938-949
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
Is this coIs this co‐‐localization of TF and GPIblocalization of TF and GPIbαα in activated platelets in activated platelets functionally relevantfunctionally relevant
Pearson Coeff 057
5 microm
GPIbα
MergeTF
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
PLATELET‐BASED MODEL OF HEMOSTASIS A PROPOSAL
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-
ACKNOWLEGMENTSACKNOWLEGMENTSOlga Panes conceived the study as main author performed the radiolabeling assays WBacutes IPacutes biotinilation and pro‐coagulant assays
Valeria Matus PhD developed all the molecular biology studies
Claudia Saacuteez PhD performed all the imaging studies
Paula Ibarra BQ thesist did the lipid raft studies
Jaime Pereira MD contributed in the design of the study discussion and critical interpretation of data and provided original ideas along the study
Antibodies against TF and GPIbα were kindly provided by Jim Morrissey Bob Montgomery and W Ruff
- Insuficiencias del Modelo
- Initiation
- Amplification
- Propagation
- Coagulation
- Blood-borne TF iquestde doacutende viene
- Platelet activation induces cell-surface immunoreactive tissue factor expression which is modulated differently by antiplatel
- 1 Conclusions
- 2 Conclusions
-