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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) *p2.5 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%

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%) 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

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 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

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 (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 57 150,000) 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**

SERF-TTP is the first prospective case-control study to investigate epidemiology, risk factors & outcomes in patients with first episode of idiopathic TTP. METHODS: Each case of TTP is identified upon referral for therapeutic plasma exchange (TPE). Exclusion criteria include organ or allogeneic stem cell transplant, anti-neoplastic therapy or malignancy. 2 age & gender-matched controls are identified for each case. Epidemiologic information, lab data & plasma samples are collected from each case. Case & control data are collected by standardized interview. Relative risk & 95% CI computed by chi-squared tests. ADAMTS13 activity & inhibitor are centrally measured on pre-TPE samples. ADAMTS13 activity is measured by FRETS-vWF73 assay (Peptides Int.). ADAMTS13 inhibitor is assessed by Technozym ADAMTS13 INH ELISA (Technoclone). RESULTS: Data is available for 67 cases & 138 controls. The median age of cases is 40, & 82% are female. Medical history & exposures are shown in Table 1. Clinical & lab characteristics vary by ADAMTS13 activity (Table 2). Low titer ADAMTS13 inhibitors are detectable in some patients with normal ADAMTS13 activity. 30 day survival of cases is 95.7%. Adverse reactions to TPE occur in 59% of patients, most commonly allergic(85%) or citrate-related reactions(65%). 14.7% had a venous access complication, 50% were catheter-related thromboses. 10% of all adverse events required an ICU admission. CONCLUSIONS: Preliminary results from the case-control study suggest that predisposing factors for the development of TTP include recent infection or connective tissue disorder. Cardiovascular disease, prior history of venous thrombosis, clopidogrel use and lower income are more common in TTP cases than controls. Patients with normal ADAMTS13 activity are more likely to present with higher platelet count, abnormal renal function and neurologic symptoms. Case & Control Characteristics Cases (n=67) Controls (n=138) Relative Risk (95% CI) Medical History Connective Tissue Disease 11.9% 2.2% 5.4 (1.5–20.0) Cardiovascular Disease 13.4% 3.6% 3.7 (1.29–10.6) Prior Venous Thrombosis 13.4% 2.9% 4.6 (1.48–14.5) Infection (prior 2 wks) 32.8% 10.1% 3.2 (1.77–5.9) Medications (prior 12 wks) Antibiotics 31.3% 17.4% 1.8 (1.08–2.99) Clopidogrel 4.5% 0.7% 6.4 (0.65–62.9) Income Level < $25,000 42% 18% 2.3 (1.47–3.6) $25,000 – 70,000 37.3% 52.9% 0.7 (0.49–0.99) > $70,000 11.9% 24.6% 0.5 (0.24–0.98) Characteristics by ADAMTS13 activity ADAMTS13 activity N ADAMTS13 inhibitor (>15 u/ml) ADAMTS13 inhibitor titer (mean) Platelets (mean) Creatinine (mean) Neurologic symptoms Survival Comparison of ADAMTS13 ≤20% vs > 20%, p-values: * 0.0028, † 0.003, ‡ 0.73, # 0.48. < 5% 18 (37.5%) 100% 80.9 22,350 1.4 44% 94% 5–20% 4 (8.3%) 100% 76.4 20,000 1.1 75% 100% > 20% 26 (54.2%) 57.7% 22.5 58,810* 4.3† 58%‡ 89.5%#

Background: Ticlopidine and clopidogrel, thienopyridine derivatives used for cerebrovascular events and coronary artery syndromes, are the first and third most common causes of drug-related TTP reported to the FDA. Ticlopine-associated TTP has been characterized as having later onset, having higher relapse rates, and being more responsive to plasmapheresis—findings associated with ADAMTS13-deficiency. Based on a limited number of ADAMTS13 measurements, Moake (NEJM 2002) and Bennett (NEJM 2000) speculated that antibody-mediated ADAMTS13-deficiency appeared to be the cause of both ticlopidine- and clopidogrel-associated TTP. Methods: Clinical and ADAMTS13 laboratory data for TTP associated with ticlopidine (n=116 and 12 cases, respectively) and clopidogrel (n= 46 and 7 cases, respectively) were reviewed. Results: Clinical Evaluation: Mortality of ticlopidine-associated TTP was 34.5%. Mortality of clopidogrel-associated TTP was 22.2%. Overall moretlaity rate was 31%. Among 162 thienopyridine-associated TTP cases, late-onset TTP (89% of the cases) occurred after > 5 days of thienopyridine use and was 4-times more common with ticlopidine than clopidogrel. Early onset TTP (11% of the cases) occurred within 5-days of thienopyridine initiation and was 4-times less common with ticlopidine than clopidogrel. Laboratory Evaluation: Among 12-ticlopidine and 7-clopidogrel-associated TTP patients, laboratory assays prior to plasmapheresis identified severe ADAMTS13 deficiency 3.3-fold more often with ticlopidine (92% versus 29%, p < 0.01) and presence of inhibitory autoantibody almost 3-fold more common with ticlopidine (83% vs 30%) than clopidogrel. Among thienopyridine-associated TTP cases, patients with severe ADAMTS13-deficiency ( Conclusions: Contrary to previously proposed mechanistic hypotheses, thienopyridine-associated TTP appears to manifest as an ADAMTS13-deficiency mediated syndrome (primarily with > 5 days of ticlopidine) that has occasional relapses and an ADAMTS13-normal syndrome (primarily with < 5 days of clopidogrel) that is not associated with relapse. Although in the same drug class, the metabolites of ticlopidine and clopidogrel are different, which may account for the distinct mechanistic pathways. Immunologically mediated ADAMTS13 -deficient TTP, like ticlopidine-associated TTP, may result from immunogenic exposures that occurred 1 to 2 weeks prior to syndrome onset. In contrast, ADAMTS13-normal TTP cases, like clopidogrel-associated TTP, may result from recent exposure that occurred within 5 days prior to TTP onset leading to direct endothelial cell injury.

Background: Plasmapheresis is the mainstay of therapy for thrombotic thrombocytopenic purpura (TTP). Due to the significant mortality associated with TTP if left untreated and the effectiveness of therapy, plasmapheresis may be instituted before the diagnosis is clearly established. Our case-control study of incident TTP allowed us to evaluate the incidence of complications in cases treated at multiple sites across the United States. Methods: The SERF-TTP study is a NHLBI-funded 15-site study to identify 300 cases of incident TTP and 600 age and gender-matched community controls. Each case of incident TTP is identified upon referral for therapeutic plasma exchange (TPE). Exclusion criteria include solid organ or allogeneic stem cell transplant, anti-neoplastic therapy, or metastatic adenocarcinoma. Epidemiologic information, laboratory data, and plasma samples are collected from both patients and controls. In addition, information is collected about volume of TPE, frequency, type of replacement fluid, duration of TPE, adverse reactions associated with TPE, and concurrent therapy. Results: To date, 11 sites across the US have IRB approval, and 50 cases have been identified. Data are available for the first 32 patients, whose mean age is 41.9 years. All patients underwent TPE, a number of which received more than one type of replacement fluid. The majority of patients received fresh frozen plasma (FFP): 60.0% FFP, 46.7% cyroprecipitate poor plasma, and 16.7% albumin. Additionally, 63.3% of patients received concurrent corticosteroid therapy. The mean number of treatment days prior to the achievement of platelet count > 150,000 per microliter was 9.7 (range 0–43 days). Overall survival was 90.0%. Adverse reactions were observed in 53.3% of patients (16/30). The most frequently observed toxicity was an allergic reaction in 40% (12/30) of patients, followed by citrate-related toxicity in 30% (9/30) of patients. No patients experienced an anaphylactic reaction. 16.7% of cases (5/30) had venous access complications, most frequently a catheter-associated thrombosis (3/5). In addition, one patient had a significant hemorrhage associated with venous access, which required treatment in intensive care. Conclusions: Preliminary data from this cohort of patients suggest that adverse reactions are commonly seen in incident TTP patients undergoing TPE; the most frequent are allergic reactions to plasma. This highlights the need for development of reliable diagnostic criteria for TTP so patients are not exposed to the risks of TPE unnecessarily.

Background: Due to the rarity of TTP (approximately 4 cases per million), epidemiologic data and correlative laboratory samples from a prospectively identified incident cohort of TTP patients are needed. Methods: SERF TTP is a NHLBI-funded 15-site study targeted to identify 300 incident TTP patients and 600 age-/gender-matched community controls. Results: From nine IRB-approved collaborating sites, 22 incident TTP cases and 17 controls have been interviewed. Data and plasma (acute and convalescent samples) are available for 19 TTP patients (Table 1). Their mean age is 42.5 years (range, 20 – 80 years), and 90% are female. Using the Rose-Eldor TTP Scoring system based on platelets, creatinine, hemoglobin, and neurologic function, 37% would be classified as severely ill (score > 4 of a maximum 8 points). Mean time from symptom onset to diagnosis was 9.2 days, median 5.5 days (range, 0 to 33 days). Severity of TTP was poorer for the 5 patients whose treatment did not begin for at least 16 days (16–33 days) versus the other 14 patients (mean Rose-Eldor score of 4.6 versus 4). Medications which had been prescribed to the patients prior to TTP onset included herbal supplements (n= 5) and hormone replacement therapy or oral contraceptives (n= 6). All patients received at least daily therapeutic plasma exchange (TPE). A platelet count >150,000 was reached at a mean of 9.4 days following TPE initiation. At 30-days follow-up, all patients were alive, although 3 had an exacerbation requiring daily TPE reinstitution. Fourteen patients (78%) experienced an adverse event, including allergic reactions to plasma (n=9), citrate-related toxicity (n=9) and venous access complications (n=2) including one with major hemorrhage requiring transfer to the intensive care unit. Conclusion: The clinical characteristics of the SERF-TTP cohort are similar to those reported from large single-site studies, although the survival rate (100% versus 71% and 83%) is higher. Only a minority of incident TTP cases in the modern era present with renal insufficiency or neurologic findings - highlighting the importance of developing reliable diagnostic laboratory testing. Clinical Characteristics at Presentation Study Platelets 2mg/dl Fever Neurologic symptoms Mortality (1) Zheng XL, Kaufman RM et al. Blood.2004; 103: 4043; (2)Vesely SK, George JN et al. Blood.2003; 102: 60. HSCT: Hematopoetic stem cell transplantation, FK-506: Tacrolimus. SERF-TTP n=19 95% 79% 32% 11% 47% 0% Washington University (1) (excluding HSCT/FK506 cases) n= 28 96% 82% 29% 32% 50% 29% Oklahoma TTP-HUS Registry (2) (ADAMTS13 < 5%) n=18 100% 100% 22% NA (Not Available) 56% 17%

Introduction: The antiplatelet agents ticlopidine and clopidogrel are thienopyridine derivatives that are commonly used for secondary prevention of cerebrovascular events, prevention of coronary artery stent thrombosis, and for treatment of acute coronary syndrome. While these chemically related agents are the first and third most common causes of drug-associated TTP respectively, clinical presentations, laboratory findings, and outcomes suggest that there may be two distinct mechanistic pathways. Methods: Clinical and laboratory data for 60 cases of ticlopidine-associated TTP and 35 cases of clopidogrel associated TTP were reviewed. Results: Type 1 thienopyridine-associated TTP, characterized by onset of disease > 5 days after drug initiation, occurred in 98% of the ticlopidine patients versus 63% of the clopidogrel patients. Laboratory studies identified severe deficiency of ADAMTS13 activity and the presence of IgG inhibitors in 7 ticlopidine (Tsai, et al.; Annals of Internal Medicine, 2000) and 2 clopidogrel (Bennett, et al.; NEJM, 2000) patients. Survival was 68% in the ticlopidine patients versus 91% in the clopidogrel patients. Type 2 thienopyridine-associated TTP, consisting of TTP onset within 5 days of drug initiation, occurred in 2% of the ticlopidine patients versus 37% of the clopidogrel patients. Laboratory studies failed to identify deficient ADAMTS13 activity or the presence of IgG inhibitors in 1 clopidogrel patient (Zheng, et al.; Blood, 2004). Survival was 0% in the ticlopidine patients versus 38% in the clopidogrel patients. Conclusion: The proposed type 1 and type 2 thienopyridine-associated TTP may be distinguished on the basis of clinical presentation, ADAMTS13 levels, and outcome. Type 1 thienopyridine-associated TTP—characterized as occurring after > 5 days of thienopyridine treatment, severely deficient ADAMTS13 activity and presence of IgG inhibitors, and high survival rates with plasmapheresis—is more likely to occur in patients receiving ticlopidine. Type 2 thienopyridine-associated TTP—characterized as occurring within 5 days of thienopyridine therapy initiation, normal ADAMTS13 activity and undetectable IgG inhibitors, and poor survival rates despite plasmapheresis—is more likely to occur in patients receiving clopidogrel. Additional confirmatory studies on larger numbers of patients are needed.