Raife, Thomas J.

Abstract Background : Many idiopathic thrombotic thrombocytopenia purpura (TTP) patients have severe deficiency of ADAMTS13, an enzyme that cleaves ultralarge von Willebrand multimers. We recently reported that thienopyridine-associated TTP is characterized by an immunologic pathway with severe ADAMTS13 deficiency and a non-immunologic pathway with higher ADAMTS13 activity levels. We now compare findings for idiopathic and thienopyridine-associated TTP patients. Methods : Clinical findings and laboratory findings were evaluated for 51 idiopathic and 39 thienopyridine-associated TTP. Results: Clinical findings were similar between idiopathic and thienopyridine-associated TTP for both severe ADAMTS13 deficient and non-deficient patients. Differences were noted in gender and age, relapse rates, and survival. Conclusion : Among TTP patients with ADAMTS13 deficiency, relapses are frequent in idiopathic TTP patients and Rituximab may be useful, while for thienopyridine-associated TTP patients spontaneous relapse are rare as long as no re-exposure occurs. Among ADAMTS13 non-deficient patients, survival is high following therapeutic plasma exchange (TPE) for idiopathic patients but not for thienopyridine-associated TTP patients. Despite similarities, idiopathic and thienopyridine associated TTP probably have different initiating factors. ADAMTS13 activity and clinical characteristics in idiopathic and thienopyridine-associated TTP Idiopathic severe ADAMTS13 deficiency (n=29) †Thienopyridine severe ADAMTS13 deficiency (n=26) Idiopathic non-severe ADAMTS13 deficiency (n=22) †Thienopyridine non-severe ADAMTS13 deficiency (n=13) *p<0.05 for comparison of severe ADAMTS13 deficiency versus non-severe ADAMTS13 deficiency. †Thienopyridine-associated TTP clinical and presenting neutralizing autoantibody data was summarized from the Journal of the American College of Cardiology publication by Bennett et al. in 2007. ‡Thienopyridine-associated TTP remission autoantibody data was summarized Tsai et al, 2002, Bennett et al, 2000, and Orimo et al, 2002, and included 3 patients. §Data from average of ADAMTS13 deficient and non-deficient cohorts. Mean Age 41 67 47 60 Sex (% female) 79% 54% 86% 39% Platelet count/mm3 (mean)* 18,978 9,269 51,409 35,000 Platelet count <20,000/mm3* 69% 96% 23% 38% Hemoglobin (mg/dl; mean) 8.5 8.3 9.0 8.4 Creatinine (mg/dl; mean)* 1.5 2.2 3.2 3.0 Creatinine >2.5 mg/dl* 11% 27% 48% 46% 30-day survival 96% 85% 90% 62% Long-term relapse* 42% 8% 0% 0% Neutralizing autoantibodies to ADAMTS13 (prior to TPE) § 79% 100% 27% 0% Neutralizing autoantibodies to ADAMTS 13 (measured at remission) § 55% 33%‡ 33% 0%

Abstract Background: Thrombotic thrombocytopenic purpura (TTP) is a rare disorder that presents with microangiopathic hemolytic anemia and thrombocytopenia, fevers, renal insufficiency and neurologic features. We reviewed clinical, laboratory, and outcome data for TTP cases with severely deficient versus non-severely deficient ADAMTS13 activity levels. Methods: Mean and median data were from the Surveillance, Epidemiology and Risk Factors for TTP (SERF-TTP) study group for idiopathic TTP cases, the Canadian Apheresis Group (CAG), and five published series (Zheng 2004, Raife 2004, Vesely 2003 (Oklahoma TTP-HUS Registry), Matsumoto 2004 (Japan Referral Center), Bennett 2007). Results: Compared to TTP cases with near-normal ADAMTS13 activity levels (n= 282), TTP cases with severe ADAMTS13-deficiency (n=185) were more likely to have severe thrombocytopenia, normal renal function and neutralizing ADAMTS13 antibodies. Severe ADAMTS13 deficient TTP cases have better overall survival after therapeutic plasma exchange (TPE) but are more likely to relapse. TTP patients with severe ADAMTS13 deficiency were primarily categorized as idiopathic or ticlopidine-associated, while TTP patients with non-severely deficient ADAMTS13 activity levels were frequently categorized as idiopathic, secondary to drugs (clopidogrel, quinine), stem cell transplantation, or cancer. Conclusions: Severe ADAMTS13 deficiency is most commonly idiopathic, has better survival following TPE, and a 35–40% spontaneous relapse rate. By contrast, non-ADAMTS13 deficient TTP cases are usually associated with an underlying disorder or external insults. Amongst this cohort, four series have 47–62% survival rates and three series, which contain mostly idiopathic cases, have 83–90% survival rates following TPE. From this, we propose that TTP may occur by three possible mechanism; ADAMTS13-deficient (antibody-mediated), an immunologic mediated pathway independent of ADAMTS13 (i.e. quinine) that is responsive to TPE, and endothelial injury related TTP that is unresponsive to TPE. Platelet count mean (x10^9/L) Creatinine mean (mg/dl) ADAMTS13 neutralizing antibodies (%) Survival % Relapse % * <15% ADAMTS13 activity cutoff Severe ADAMTS13 Deficiency (<10–15%) SERF-TTP (n=30) 19 1.3 83 97 41 Zheng (n=16) 19 1.6 44 81 38 Bennett (n=26) 15 85 Oklahoma (n=18) 12 1.8 94 81 38 Raife (n=50) * 13 1.2 92 35 Japan (n=34) 35 91 Canada (n=11) 16 2.4 82 Not Severely Deficient ADAMTS13 Activity (> 15%) SERF-TTP (n=22) 57 3.9 35 90 0 Raife (n=57) * 44 2.7 83 9 Canada (n=17) 57 4.1 88 Zheng (n=13) 40 3.0 0 54 Bennett (n=13) 62 Japan (n=66) 9 62 Oklahoma (n=94 ) 23 47 3

Abstract Thrombotic thrombocytopenic purpura (TTP) is a microvascular occlusive disorder characterized by systemic aggregation of platelets, thrombocytopenia, and mechanical injury to erythrocytes. Between 30% and 80% of TTP cases are associated with ADAMTS13 deficiency. Thienopyridine-derivative anti-platelet agents, ticlopidine and clopidogrel, are the drugs most commonly associated with TTP. The structures differ only by a carboxymethyl side-chain and have no common metabolites. Since 2002, our R01 research project has focused on evaluating thienopyridine-associated TTP. Herein, we present the final results. Clinical and laboratory data were obtained from case reports, the FDA’s MedWatch program, a Japanese national reference laboratory for ADAMTS13 assays, and apheresis centers at Duke University, University of North Carolina, Northwestern University, and the Mayo Clinic. Epidemiologic data for rate estimation for thienopyridine-associated TTP among persons who receive cardiac stents were obtained from international cardiology laboratories. Pharmacovigilance information was obtained from package inserts for the drugs. Most thienopyridine-associated TTP cases are associated with two weeks or more of ticlopidine rather than clopidogrel, are immune-mediated involving neutralizing antibodies to ADAMTS13, resolve with therapeutic plasma exchange (TPE), and have spontaneous relapses. Less frequently, cases are associated with clopidogrel, occur within days of drug initiation, may be a direct result of endothelial cell damage, are less responsive to TPE, and are less likely to recur. Thienopyridine-associated TTP patients with severe deficiency of ADAMTS13 activity have a different profile than those with normal ADAMTS13 levels. Among thienopyridine-associated TTP patients who have ADAMTS13 deficiency, TPE is usually performed for a few days and patients recover without detectable organ damage. In contrast, among thienopyridine-associated TTP patients who do not have ADAMTS13 deficiency, several weeks of TPE is required for recovery, and 30% mortality rates have been reported. Despite similar chemical structures, ticlopidine- and clopidogrel-associated TTP probably occur by different mechanisms and have different clinical presentations and expected outcomes. Clinical Characteristics Onset Platelet Count <20,000 (%) Creatinine ≤2 mg/dl (%) Neurologic Symptoms (%) Mortality With TPE/Without TPE (%) Relapse *p < 0.05 ticlopidine 10%* <2 weeks; 90% 2-12 weeks 84* 28* 28 15/44 Occasional clopidogrel 74%*≤2 weeks; 26% > 2 weeks 60* 55* 32 28/33 Rare Epidemiology, Pharmacovigilance & Basic Science Incidence (cases per treated patients) Safety ADAMTS13 Activity (%) ADAMTS13 Antibodies (%) ticlopidine 1:1,600 “Black Box” warning <10* 90* clopidogrel 1:100,000 package insert warning 50–100* 10*

Abstract Background: The Surveillance, Epidemiology & Risk Factors for TTP (SERF-TTP) study is the largest prospective cohort of idiopathic TTP cases to date. Methods: Patients with first episode of idiopathic TTP were enrolled at 11 sites in the US. Exclusion criteria include solid organ or allogeneic stem cell transplant, anti-neoplastic therapy or malignancy. All patients underwent therapeutic plasma exchange (TPE). Remission is defined as platelet count >150,000/mm3. ADAMTS13 activity was measured by fluorescence resonance energy transfer assay (FRETS-vWF73, Peptides Int.). ADAMTS13 inhibitor was assessed by ELISA (Technozym ADAMTS13 INH ELISA, Technoclone) and functional inhibition of normal ADAMTS13 activity (modified FRETS). Differences between the groups was evaluated by Chi-square test, t-test or Fisher’s exact test. Results: Complete data is available for 57 cases. 84% were female & median age was 42. ADAMTS13 was severely deficient (<10%) in 53%, moderate (10–50%) in 8%, & normal (>50%) in 39%. Adverse events were frequent & most commonly include citrate toxicity and allergic reactions. Long-term followup was available for 56 patients, and overall relapse rate was 25% with median time to relapse of 11 months. The relapse rate was 41% in patients with severe ADAMST13 deficiency & 0% in patients with normal ADAMTS13 activity (p=0.0077). Conclusions: Severe ADAMTS13 deficiency does not define all cases of idiopathic TTP, yet is associated with a unique syndrome characterized by severe thrombocytopenia, normal renal function, presence of ADAMTS13 neutralizing autoantibodies & high risk of relapse. Non-ADAMTS13 deficient idiopathic TTP has clinical features similar to thrombotic microangiopathies associated with stem cell transplant or drugs such as quinine, yet has a better than expected overall survival after TPE. There is likely an alternate disease mechanism for this cohort, possibly immunologic, which may be responsive to TPE and warrants further study. ADAMTS13 < 10% n=30 ADAMTS13 > 50% n=22 p-value * t-test, ** Fisher’s Exact Test, # Chi-Square Test Presenting Labs Hemoglobin (mean) 8.5 9.2 0.22* Platelet count (mean x10^9/L) 19 57 <0.0005* Serum creatinine (mean) 1.33 3.9 0.0012* ADAMTS13 antibody (neutralizing & non-neutralizing) % 100 63.6 0.0004** ADAMTS13 neutralizing antibody % 83 35 .0003# Presenting Symptoms Neurologic symptoms present 45 52.4 0.23# Diarrhea 23 30.4 0.79# Infection 40 43.5 0.79# Past Medical History Connective Tissue Disease % 10 8.5 0.28# Family history of TTP % 0 4.6 0.11# Remission Labs Hemoglobin (mean) 12.2 8.5 0.16* Platelet count (mean x10^9/L)) 169 152 0.15* Serum creatinine (mean) 1.02 3.7 0.0009* Outcomes Therapy-related Adverse events % 71 55 0.24# Time (days) to remission (plt > 150,000) 9.7 9.2 0.89* Early remission (< 8 days) % 73 57 0.20# 30 day Exacerbation rate % 35 28 0.63# 30 day Survival % 96.6 90.0 0.56** Long-term Relapse % 41 0 0.0077**

Abstract Abstract 2203 Background: Thrombotic thrombocytopenic purpura (TTP) is frequently characterized by severe ADAMTS13 deficiency, the cause of which is unknown. An important exception is ticlopidine-associated TTP (tc-TTP), the most common drug-induced TTP syndrome. In 1998, a possible association of TTP with ticlopidine was reported. In 1999, two additional series reported this association. In 2000, a fourth study reported severe A DAMTS13 deficiency among six of seven tc-TTP persons- suggesting a causal pathway. All of these reports were from the United States. Recently, we reported characteristics of 186 acquired idiopathic (ai)- TTP patients in Japan with severe ADAMTS13 deficiency, noting that clinical and laboratory findings for ai-TTP patients in Japan differed from those for cohorts of ai-TTP patients in Europe and North America- raising concern that TTP findings vary by region of the world. (PLOS One 2011) We now on report clinical and laboratory characteristics of a cohort of TTP patients from Japan with tc-TTP, and compare these findings to three cohorts of tc-TTP in the United States and one cohort of ai-TTP patients from Japan. Methods: We queried a database of thrombotic microangiopathy patients identified from a national TTP referral laboratory in Japan of cases identified between 1998 and 2008. Severe ADAMTS13 deficiency was characterized by activity levels < 5%. All tc-TTP patients and 186 of 911 ai-TTP patients in the database had severe ADAMTS13 deficiency and first onset of TTP. Comparisons were made to tc-TTP patients reported previously from the United States. Results: Characteristics of ai- TTP with severe ADAMTS13 deficiency in Japan and tc-TTP in Japan and US Conclusions: These data from Japan validate insights about tc-TTP initially proposed in 1998, 1999, and 2000 in the United States. Ticlopidine is a likely cause of TTP, the mechanism is via a cross-reactive antibody to ADAMTS13:AC resulting in formation of an ADAMTS13:INH, and therapeutic plasma exchange is necessary for treatment. Disclosures: Ortel: Eisai: Research Funding; Glaxo SmithKline: Research Funding; Pfizer: Research Funding; Instrumentation Laboratory, Inc: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.