adamts13 max smith

8/20/2019 Adamts13 Max Smith

1/63

“What is ADAMTS13 Anyway?”

Maxwell Smith, MD

August 19th, 2005


2/63

Objectives

• Review the pathology, epidemiology, differentialdiagnosis, and treatment of ThromboticThrombocytopenic Purpura (TTP) using a casepresentation

• Describe the discovery of ADAMTS13 (vonWillebrand Factor Cleaving Protease) and its rolein TTP

• Introduce selected testing methods for vWF-CPand evaluate their use in the diagnosis andmanagement of TTP


3/63

35 year-old Hispanic Female

• Presents with easy bruising and abdominalpain

• Multiple bruises had developed “all over”without traumatic history

• Multiple small red spots as well

• No neurologic complaints• 1 week prior had URI symptoms and

diarrhea


4/63

Physical Exam

• Vitals: Temp 38.5, HR 94, BP 121/75

• Active gingival and mucosal bleeding

• Innumerable ecchymoses over all

extremities and trunk

• No neurologic abnormalities

• Scattered petechiae on BUE and BLE

• Cervical lymphadenopathy


5/63

Lab Studies

• CBC: WBC 12.8, HCT 30, PLTC 12

• BUN/Cr: 14 / 0.9

• CoAg: PT 13.2, PTT 40.9, d-dimer 3.91• T-bili: 2.5 (2.4 unconjugated)

• LDH 1522

• Peripheral smear

– Thrombocytopenia– 1+ schistocytes

• Direct Coombs neg.

• Drug screen neg.


6/63

Brief Differential Diagnosis

• TTP vs. Hemolytic uremic syndrome vs. ITP

• TTP– Classic pentad

– Patients often have only 3/5

• HUS– Lack of neurologic defects

– “Prominence” of renal compromise

• ITP– Isolated thrombocytopenia with no other clinical findings - A

diagnosis of exclusion

– No anemia, fever, neuro, or renal signs/symptoms


7/63

TTP - Classic pentad

• Fever

• Thrombocytopenia

• Transient neurologic dysfunction

• Microangiopathic hemolytic anemia

• Renal failure


8/63

TTP Pathology

• Unique composition of microvascular thrombi

– Platelet rich

– vWF rich– Fibrin poor (lack of involvement of the traditional

clotting cascade)

• Sheer forces lead to fragmentation of the RBC -

hemolytic anemia with schistocytes

• Vascular compromise leads to end organ

dysfunction (kidneys, brain, etc.)


9/63

TTP - Epidemiology

• Classic history is sudden onset of symptoms

in an otherwise health adult

• Propensity for females of child bearing age


10/63

TTP - Treatment

• Plasma exchange

– Started in 1970’s

– Changed prognosis from >90% mortality to


11/63

“What is ADAMTS13 Anyway?”

• Moake et al., 1982

– In 4 patients with chronic TTP, large multimers

of vWF were identified in their serum

– Multimers were similar to those found in media

surrounding in vitro endothelial cells

suggesting vascular cells as a possible source– Possible failure of cleavage of the vWF

multimer was causing TTP


12/63

ADAMTS13 discovery

• 1996 - 2 groups identified a 300 kD

metalloprotease which cleaved the vWF

multimers at a Tyr-Met bond– Required divalent cations (inhibited by calcium

chelating agents)

– Kinetics were slow in undisturbed plasma– Mild denaturation of the vWF protein or fluid

shear stress accelerated the reaction


13/63

ADAMTS13 discovery cont.

• Levy et al., 2001

• Mapped the gene for the metalloprotease to

chromosome 9q34 with linkage analysis• Identified a new member of the ADAMTS

family of zinc metalloproteinases,

ADAMTS13• Identified 12 mutations in patients with

hereditary TTP clinical picture


14/63

vWF-cleaving protease (ADAMTS13)

Tyr-Met AA bond in vWF

Receptor for GP Ib on the platelets

Platelet

Von Willebrand Factor Cleaving Protease, vWF, and Platelets

Under Normal Conditions


15/63

vWF and Platelets When Von Willebrand Factor Cleaving

Protease is Absent or Deficient


16/63

vWF and Platelets When Von Willebrand Factor Cleaving

Protease is Absent or Deficient, Cont.


17/63

Back to Our Patient

• Heme/Onc diagnosed TTP clinically

• Sample drawn for vWF-CP testing and sent

to the Blood Center of Wisconsin

• 7 rounds of plasma exchange over 4-5 days

• Patient discharged home with normal

platelet count

• Lab test pending


18/63

Laboratory Testing for vWF-CP

• Genomic Studies– Limited use unless documented family history of TTP

like illness (FISH analysis for multiple known geneticmutations in the ADAMTS13 gene)

• Activity & Inhibitor Studies– Wide variety of methods currently in use

– Initial methods required laboratory and personnelexpertise

– Subsequent methods have decreased turnaround timeand complexity


19/63

Flaws in current vWF-CP Testing

• The test result is invariably compared to thecurrent gold standard for TTP diagnosis -

clinical (universally accepted to be difficultand often incorrect) - misclassification bias

• Most measure enzyme activity indirectly

• Most carry out enzymatic reaction in nonphysiologic conditions

• No standardization of methods


20/63

Questions to be Answered from

the Literature• 1. What test is our send out lab using and

does it work?

• 2. How well do the various testing methods

compare with each other?

• 3. How has this specific test (or similar

tests) been evaluated and how well has itperformed?


21/63

• 1. What test is our send out lab using and

does it work?


22/63

Gerritsen et al., 1999

• Assay of von Willebrand Factor (vWF)-

cleaving Protease Based on Decreased

Collagen Binding Affinity of DegradedvWF

• Developed a simple activity and inhibitor

assay for vWF-CP• Evaluated the test in 40 patients


23/63


• Activity Assay– Deactivate donor plasma vWF-CP with EDTA

– Mix donor and patient plasma

– If vWF-CP is present and functional in the patient

sample, it will cleave vWF-multimers in the donorplasma (more vWF-CP, more vWF monomers, less vWFmultimers)

– Add solution to plates coated with human type IIIcollagen (preferentially binds vWF multimers)

– Quantify the collagen bound vWF multimers with a anti-vWF multimer peroxidase labeled antibody

– Measure absorbance at 492nm

– Calibration curve done with serially diluted donor

plasma samples


24/63


• Inhibitor assay

– Add non-deactivated donor plasma with patient

sample in 1:1 ratio• If antibodies to vWF-CP are present in the patient

sample, they will bind and deactivate vWF-CP from

the donor plasma

– Perform same test as previously described


25/63


• Plasma sample selection

– Based on clinical findings (classic pentad) and

a history of relatives with a similar condition• 10 “normal” control patients

• 10 with familial TTP

• 11 with acquired TTP

• 9 with HUS


26/63


N 10 10 11 9 10 11


27/63


• Conclusions

– vWF-CP activity can be used to distinguish

TTP from HUS– The presence of a vWF-CP inhibitor can further

differentiate acquired from hereditary TTP

– The collagen binding assay is sensitive andspecific


28/63


• Study deficiencies

– Low sample number (40)

– Misclassification bias• Diagnosis of TTP

• Definition of familial


29/63

• 2. How well do the various testing methods

compare with each other?


30/63

Studt et al., 2003

• Measurement of von Willebrand factor-

cleaving protease (ADAMTS-13) activity in

plasma: a multicenter comparison ofdifferent assay methods


31/63

Studt et al., 2003

• Methods

– Identical aliquots from 30 different patients with

acquired TTP, hereditary TTP, and “other” conditionswere sent to 5 different laboratories

– Each lab used its standard testing method for activity

and presence of inhibitor

• (1) Immunoblot assay

• (2) Residual collagen binding assays

• (1) Residual ristocetin cofactor activity assay

• (1) Immunoradiometric assay


32/63

Studt et al., 2003• Results


33/63

Studt et al., 2003•

Results Cont.


34/63

Studt et al., 2003

• Conclusion

– In general, correlation fairly good [Spearman

rank order correlation coefficient = 0.89 - 0.97(p


35/63

Studt et al., 2003

• Problems

– Dose not address the accuracy or

reproducibility of the labs (each sample wasonly tested once)

– Poor correlation of data at the higher activity

level compensated for correlation at the lower

activity levels


36/63

Tripodi et al., 2004

• Measurement of von Willebrand factor

cleaving protease (ADAMTS13): results of

an international collaborative studyinvolving 11 methods testing the same set

of coded plasmas


37/63


• Method

– Normal plasma (100% activity) and plasma

from a patient with familial TTP (0% activity)were mixed to have ADAMTS activity, by

volume, of 0%, 10%, 20%, 40%, 80%, and

100%


38/63


• Results

– Linearity (expected vs. observed) = from 0.98

to 0.39 (1 = perfect linearity)– Reproducibility = from


39/63


• Conclusion

– Best methods included measuring vWFCP by

ristocetin cofactor, residual collagen binding,and immunoblotting

– Varied inter-laboratory agreement


40/63

• 3. How has this specific test (or similar

tests) been evaluated and how well has it

performed?


41/63

Furlan et al., 1998

• Von Willebrand Factor-Cleaving Protease

in Thrombotic Thrombocytopenic Purpura

and the Hemolytic -Uremic Syndrome


42/63

Furlan et al., 1998

• Methods

– Plasma from patients with a clinical diagnosis of TTP

or HUS along with a worksheet containing clinical and

laboratory data was sent for study (selection bias)

– Patients were classified as TTP, acute or in remission

and as HUS, acute or in remission by the PI without

knowledge of the vWFCP testing (based on the work-

sheet)

– vWFCP testing using an immunoabsorbent assay


43/63

Furlan et al., 1998• Results


44/63

Furlan et al., 1998• Results Cont.


45/63

Furlan et al., 1998

• Conclusion

– Nearly absent levels of vWFCP is a sensitive

test for acute TTP (low false negative)– Plasma from patients with non-familial TTP

tends to have a vWFCP inhibitor while plasma

from those with familial TTP does not


46/63

Furlan et al., 1998

• Problems

– Test bias

– Selection bias

– No “normal” patients included

– No referred patients were excluded

– Gold standard (PI interpretation of work sheet)- misclassification bias


47/63

Bianchi et al., 2002

• Von Willebrand factor-cleaving protease

(ADAMTS13) in thrombocytopenic

disorders: a severely deficient activity isspecific for thrombotic thrombocytopenic

purpura


48/63


• Methods– 68 patients with thrombocytopenia (


49/63

Bianchi et al., 2002• Results

Only 18% had levels


50/63


• Conclusion

– These results along with prior publications

indicate that very low levels (


51/63


• Problems

– Test bias

– Misclassification bias

– Did not include normal patients or patients with

TTP in current study


52/63

Peyvandi et al., 2004

• Von Willebrand factor cleaving protease

(ADAMTS13) and ADAMTS13

neutralizing antibodies in 100 patients withTTP


53/63

Peyvandi et al., 2004• Methods

– 3 of the following present:

thrombocytopenia,

hemolytic anemia,

increased LDH, and

neurologic symptoms– Residual collagen binding

assay used for activity and

inhibitor levels

– Low ADAMTS13 activity

was


54/63

Peyvandi et al., 2004• Results

2/15 patients with


55/63


• Conclusions

– ADAMTS13 activity deficiency, regardless of

the cutoff is not exclusively diagnostic for TTPin patients with solid clinical evidence of TTP

– May be other mechanisms involved in the

inhibition of ADAMTS13 activity or other

pathways involved


56/63


• Problems

– Referral bias

– Misclassification bias


57/63

Back to Our Patient

• Pre-plasmapheresis sample was sent to the

Blood Center of Wisconsin for vWF-CP

testing using the Gerritsen method(approximately 4-5 day TAT)

– ADAMTS13 Activity = 60%)

– ADAMTS13 Inhibitor = 2 IU (RR


58/63

Will These Test Results Help

Our Patient?• TAT requires presumptive diagnosis be made and

treatment began prior to test results

• Supports the clinical impression of acquired TTP• Will not be helpful in monitoring treatment or

during remission

• Patient was treated appropriately with

plasmapheresis and recovered well, all without

test results


59/63

Conclusion

• Review the pathology, epidemiology, differentialdiagnosis, and treatment of ThromboticThrombocytopenic Purpura (TTP) using a case

presentation• Describe the discovery of ADAMTS13 (von

Willebrand Factor Cleaving Protease) and its rolein TTP

• Introduce selected testing methods for vWF-CPand evaluate their use in the diagnosis andmanagement of TTP


60/63

Closing Thoughts

• Sensitivity and specificity of vWF-CP activity maybe more closely related to the ability of clinicians todetermine between the various causes of

thrombocytopenia, rather than being specific to TTP• As with many tests, the initial reports of a “perfect”

test have not been substantiated (often, with muchless fanfare)

• Testing for vWF-CP needs further development inorder to have a positive impact on the managementof thrombocytopenic patients


61/63

References


62/63

References• Bianchi V. et al. Von Willebrand factor cleaving protease (ADAMTS13) in

thrombocytopenic disorders: a severely deficient activity is specific for TTP. Blood2002; 100; 710-713.

• Blood Center of Wisconsin. ADAMTS 13 Activity and Inhibitor, June 2005.

• Furlan M. et al. Von Willebrand factor cleaving protease in TTP and HUS. NEJM;1998; 339; 1578-84.

• Gerritsen H. et al. Assay of von Willebrand factor cleaving protease based on decreasedcollagen binding affinity of degraded vWF. Thrombosis and Haemostasis1999; 82;1386-9.

• Mannucci P. TTP: A simpler diagnosis at last? Thrombosis and Haemostasis 1999; 82;1380-1.

• Peyvandi F. et al. Von Willebrand factor cleaving protease (ADAMTS13) andADAMTS13 neutralizing antibodies in 100 patients with TTP. British Journal ofHaematology 2004; 127; 433-439

• Sadler J. A new name in thrombosis, ADAMTS13. PNAS 2002; 99; 11552-11554.

• Studt j. et al. Measurement of von Willebrand factor cleaving protease (ADAMTS13)

activity in plasma: a multicenter comparison of different methods. Journal ofThrombosis and Haemostasis 2003; 1; 1882-1887.

• Tripodi A. et al. Measurement of von Willebrand factor cleaving protease(ADAMTS13): results of an international collaborative study involving 11 methodstesting the same set of coded plasmas. Journal of Thrombosis and Haemostasis 2004; 2;1601.


63/63

adamts13 max smith

Documents