1. “What is ADAMTS13 Anyway?” Maxwell Smith, MD August 19th, 2005

2. 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

3. 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

4. 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. 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. 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. TTP - Classic pentad Fever Thrombocytopenia Transient neurologic dysfunction Microangiopathic hemolytic anemia Renal failure

8. 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. TTP - Epidemiology Classic history is sudden onset of symptoms in an otherwise health adult Propensity for females of child bearing age

10. TTP - Treatment Plasma exchange –Started in 1970’s –Changed prognosis from >90% mortality to <25% mortality –Effectiveness related to the proposed etiology of TTP

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

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

17. 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. 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

19. 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

20. 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?

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

22. 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

23. 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

24. 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

25. 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

26. Gerritsen et al., 1999 N 10 10 11 9 10 11

27. 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

28. Gerritsen et al., 1999 Study deficiencies –Low sample number (40) –Misclassification bias Diagnosis of TTP Definition of familial

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

30. Studt et al., 2003 Measurement of von Willebrand factor- cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods

31. 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

32. Studt et al., 2003 Results

33. Studt et al., 2003 Results Cont.

34. 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

35. 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

36. 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

37. 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%

38. 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

39. Tripodi et al., 2004 Conclusion –Best methods included measuring vWFCP by ristocetin cofactor, residual collagen binding, and immunoblotting –Varied inter-laboratory agreement

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

41. Furlan et al., 1998 Von Willebrand Factor-Cleaving Protease in Thrombotic Thrombocytopenic Purpura and the Hemolytic -Uremic Syndrome

42. 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. Furlan et al., 1998 Results

44. Furlan et al., 1998 Results Cont.

45. 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. 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. Bianchi et al., 2002 Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura

48. 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

49. Bianchi et al., 2002 Results Only 18% had levels <30% 10% is lowest level

50. 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

51. Bianchi et al., 2002 Problems –Test bias –Misclassification bias –Did not include normal patients or patients with TTP in current study

52. Peyvandi et al., 2004 Von Willebrand factor cleaving protease (ADAMTS13) and ADAMTS13 neutralizing antibodies in 100 patients with TTP

53. 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%

54. Peyvandi et al., 2004 Results 2/15 patients with <20% ADAMTS13 activity and no inhibiting antibodies had mutations in the ADAMTS13 gene

55. 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

56. Peyvandi et al., 2004 Problems –Referral bias –Misclassification bias

57. 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 <0.5IU) 1 inhibitor unit will decrease the expected enzyme activity by 50%

58. 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. 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

60. 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

62. 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. 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.