“What is ADAMTS13 Anyway?”

Maxwell Smith, MD

August 19th, 2005

Objectives

• Review the pathology, epidemiology, differential diagnosis, and treatment of Thrombotic Thrombocytopenic Purpura (TTP) using a case presentation

• Describe the discovery of ADAMTS13 (von Willebrand Factor Cleaving Protease) and its role in TTP

• Introduce selected testing methods for vWF-CP and evaluate their use in the diagnosis and management of TTP

35 year-old Hispanic Female

• Presents with easy bruising and abdominal pain

• 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

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

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.

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

TTP - Classic pentad

• Fever

• Thrombocytopenia

• Transient neurologic dysfunction

• Microangiopathic hemolytic anemia

• Renal failure

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.)

TTP - Epidemiology

• Classic history is sudden onset of symptoms in an otherwise health adult

• Propensity for females of child bearing age

TTP - Treatment

• Plasma exchange– Started in 1970’s– Changed prognosis from >90% mortality to

<25% mortality– Effectiveness related to the proposed etiology

of TTP

“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

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

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

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

vWF and Platelets When Von Willebrand Factor Cleaving Protease is Absent or Deficient

vWF and Platelets When Von Willebrand Factor Cleaving Protease is Absent or Deficient, Cont.

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

Laboratory Testing for vWF-CP

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

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

• Activity & Inhibitor Studies– Wide variety of methods currently in use– Initial methods required laboratory and personnel

expertise– Subsequent methods have decreased turnaround time

and complexity

Flaws in current vWF-CP Testing

• The test result is invariably compared to the current gold standard for TTP diagnosis - clinical (universally accepted to be difficult and often incorrect) - misclassification bias

• Most measure enzyme activity indirectly• Most carry out enzymatic reaction in non

physiologic conditions• No standardization of methods

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 it performed?

• 1. What test is our send out lab using and does it work?

Gerritsen et al., 1999

• Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF

• Developed a simple activity and inhibitor assay for vWF-CP

• Evaluated the test in 40 patients

Gerritsen et al., 1999• 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 donor plasma (more vWF-CP, more vWF monomers, less vWF multimers)

– Add solution to plates coated with human type III collagen (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

Gerritsen et al., 1999

• 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

Gerritsen et al., 1999

• 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

Gerritsen et al., 1999

N 10 10 11 9 10 11

Gerritsen et al., 1999

• 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 and

specific

Gerritsen et al., 1999

• Study deficiencies– Low sample number (40)– Misclassification bias

• Diagnosis of TTP

• Definition of familial

• 2. How well do the various testing methods compare with each other?

Studt et al., 2003

• Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods

Studt et al., 2003

• Methods– Identical aliquots from 30 different patients with

acquired TTP, hereditary TTP, and “other” conditions were 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

Studt et al., 2003• Results

Studt et al., 2003• Results Cont.

Studt et al., 2003

• Conclusion– In general, correlation fairly good [Spearman

rank order correlation coefficient = 0.89 - 0.97 (p<0.001)]

– Deviations were more common in the collagen binding assay suggesting it is more delicate

Studt et al., 2003

• Problems– Dose not address the accuracy or

reproducibility of the labs (each sample was only tested once)

– Poor correlation of data at the higher activity level compensated for correlation at the lower activity levels

Tripodi et al., 2004

• Measurement of von Willebrand factor cleaving protease (ADAMTS13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas

Tripodi et al., 2004

• 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%

Tripodi et al., 2004

• Results– Linearity (expected vs. observed) = from 0.98

to 0.39 (1 = perfect linearity)– Reproducibility = from <10% to 83%

(coefficient of variation)– Better correlation between the very low and

high ADAMTS13 levels

Tripodi et al., 2004

• Conclusion– Best methods included measuring vWFCP by

ristocetin cofactor, residual collagen binding, and immunoblotting

– Varied inter-laboratory agreement

• 3. How has this specific test (or similar tests) been evaluated and how well has it performed?

Furlan et al., 1998

• Von Willebrand Factor-Cleaving Protease in Thrombotic Thrombocytopenic Purpura and the Hemolytic -Uremic Syndrome

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

Furlan et al., 1998• Results

Furlan et al., 1998• Results Cont.

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

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

Bianchi et al., 2002

• Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura

Bianchi et al., 2002

• Methods– 68 patients with thrombocytopenia (<140 K) recruited for

study with the following clinical diagnoses• Sepsis (17)• HIT (16)• Osteomyelofibrosis (3)• Myelodysplastic syndrome (4)• ITP(10)• Acute leukemia (6)• Severe aplastic anemia (2)• Miscellaneous (10)

– ADAMTS13 activity and inhibitor measurement with immunoblotting method

Bianchi et al., 2002• Results

Only 18% had levels <30%

10% is lowest level

Bianchi et al., 2002

• Conclusion– These results along with prior publications

indicate that very low levels (<5% activity) of ADAMT13 activity is very specific for TTP

Bianchi et al., 2002

• Problems– Test bias– Misclassification bias– Did not include normal patients or patients with

TTP in current study

Peyvandi et al., 2004

• Von Willebrand factor cleaving protease (ADAMTS13) and ADAMTS13 neutralizing antibodies in 100 patients with TTP

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 <46%

Peyvandi et al., 2004• Results

2/15 patients with <20% ADAMTS13 activity and no inhibiting antibodies had mutations in the ADAMTS13 gene

Peyvandi et al., 2004

• Conclusions– ADAMTS13 activity deficiency, regardless of

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

– May be other mechanisms involved in the inhibition of ADAMTS13 activity or other pathways involved

Peyvandi et al., 2004

• Problems– Referral bias– Misclassification bias

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 = <4% (RR>60%)– ADAMTS13 Inhibitor = 2 IU (RR <0.5IU)

• 1 inhibitor unit will decrease the expected enzyme activity by 50%

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

Conclusion

• Review the pathology, epidemiology, differential diagnosis, and treatment of Thrombotic Thrombocytopenic Purpura (TTP) using a case presentation

• Describe the discovery of ADAMTS13 (von Willebrand Factor Cleaving Protease) and its role in TTP

• Introduce selected testing methods for vWF-CP and evaluate their use in the diagnosis and management of TTP

Closing Thoughts

• Sensitivity and specificity of vWF-CP activity may be more closely related to the ability of clinicians to determine 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 much less fanfare)

• Testing for vWF-CP needs further development in order to have a positive impact on the management of thrombocytopenic patients

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

thrombocytopenic disorders: a severely deficient activity is specific for TTP. Blood 2002; 100; 710-713.

• Blood Center of Wisconsin. ADAMTS 13 Activity and Inhibitor, June 2005. <www.bcw.edu>

• 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 decreased collagen 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) and ADAMTS13 neutralizing antibodies in 100 patients with TTP. British Journal of Haematology 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 of Thrombosis 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 methods testing the same set of coded plasmas. Journal of Thrombosis and Haemostasis 2004; 2; 1601.