ALBERT

Description: Background: The patients with severe aortic-valve stenosis (AS) are often complicated with bleeding episodes. The association between AS and gastrointestinal bleeding due to angiodysplasia is reported as Heyde's syndrome, which is categorized as one of the acquired von Willebrand disease (AVWD) in cardiovascular disorders. An international survey has shown that Type 2A is the common subtype of AVWD in the patients with AS. AVWD Type 2A is characterized by impaired platelet-dependent VWF function caused by marked decrease or absence of the most hemostatically active HMW-VWFM. In the patients with AS, a significant correlation between the increased high shear stress and loss of HMW-VWFM in vivo. Further, the absence of HMW-VWFM and bleeding tendency are normalized after valve replacement. These results suggest that enhanced proteolysis of von Willebrand factor (VWF) as it passes through the stenotic valve may induce the loss of HMW-VWFM because high shear stress can induce structure changes in VWF, which is sensitive form to the VWF cleaving protease, termed ADAMTS13. Here, we performed investigation of plasma levels of VWF antigen (VWF:Ag), ADAMTS13 activity (ADAMTS13:AC), and platelet thrombus formation in the patients with AS by valve replacement to confirm the pathophysiological mechanism of this rare disease. Patients and Methods: Ten consecutive patients who underwent aortic valve replacement for AS in Nara Medical University Hospital were enrolled in this study. The severity of AS was judged by the American Heart Association guideline. All patients had no bleeding history and received bovine tissue valves replacement followed by administration of warfarin and/or anti-platelet agents for prevention of thrombosis a week after surgery. We collected a series of blood samples from these patients before and day1, 8, 15, 22 after valve replacement. Excluding one patient who developed critical cardiac failure just after valve replacement, 9 patients were eventually evaluated by analyses of VWF:Ag, VWF multimers, ADAMTS13:AC, and mural thrombus formation using flow chamber system. VWF:Ag was measured by sandwich ELISA using a rabbit anti-human VWF polyclonal antiserum. Analysis of VWF multimers was performed according to the method of Ruggeri and Zimmerman. ADAMTS13:AC was measured by a chromogenic ADAMTS13-act-ELISA. Platelet thrombus formation was evaluated by thrombus generation under a high shear stress in a parallel plate flow chamber system. Briefly, whole blood anti-coagulated with argatroban was incubated with the fluorescent dye DiOC6 (1uM), and these samples containing DiOC6 -labeled platelets were perfused for 7 min over a type I collagen-coated glass surface under a high shear rate (1500 s-1). The DiOC6 fluorescence corresponding to the platelets was examined at an excitation wavelength of 488 nm with a barrier filter at 500 nm. The percentage of the area covered by adhering platelets (surface coverage) and each thrombus volume were evaluated. Results: Plasma levels of VWF:Ag before surgery were 78.1 % (median) and those on day 1, 8, 15, 22 after surgery were 130, 224, 155, and 134 %, respectively (Fig 1). Conversely, these levels of ADAMTS13:AC were 50.5, 35.5, 25.5, 25.1, and 30.3 %, respectively (Fig 2). The ratio of VWF:Ag/ADAMTS13:AC at before and day 1, 8, 15, 22 after surgery were 1.6, 4.5, 8.1, 6.1, and 4.1, respectively. In VWF multimer analysis, we found the obvious defect of HMW-VWFM in 7 of 9 patients before surgery, who were diagnosed with severe AS. The remaining two patients had moderate AS with the slight defect of HMW-VWFM. These defects were improved within 14 days after surgery. In platelet thrombus formation, the amount of thrombus volumes significantly increased at day 8, 15, and 22 after compared with before surgery (Fig 3). Conclusion: The dramatic recovery of platelet thrombus formation was observed in the patients with AS by valve replacement. The rapid increment of VWF and normalization of VWFM pattern, together with reduction of ADAMTS13 after valve replacement suggested heightened proteolysis of VWF by ADAMTS13 under high shear stress would be a major cause of this unique bleeding complication. The highest ratio of VWF:Ag/ADAMTS13:AC at day 8 after surgery might imply the necessity of blockade of heightened VWF function with anti-platelet agents. Figure 1 Figure 1. Disclosures Matsumoto: Alfresa Pharma Corporation: Patents & Royalties. Fujimura:Alfresa Pharma Corporation: Patents & Royalties.

Description: 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 % *

Description: 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

Description: Abstract 3182 Poster Board III-119 Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity. Disclosures No relevant conflicts of interest to declare.

Description: Background/aims: Deficiency of plasma ADAMTS13 activity (:act) accumulates unusually large von Willebrand factor multimer (UL-VWFM) in circulation that might induce platelet thrombi formation. We demonstrated that hepatic stellate cells are major ADAMTS13-producing cells in human liver using in situ hybridization and immunohistochemistry (Blood, 2005, 106:922), and a decreased plasma ADAMTS13:act in patients with cirrhotic biliary atresia can be fully restored after living-related liver transplantation (Blood2000, 96:636a). Taken these findings together, ADAMTS13 may play a role on the regulation of sinusoidal microcirculation in the liver. We, therefore, investigated the relationship of ADAMTS13 and its substrate (UL-VWFM) to clinical features in patients with chronic liver diseases. Methods: Plasma levels of ADAMTS13:act, ADAMTS13 antigen (:ag) and VWF antigen (VWF:ag) were determined in 33 patients with chronic hepatitis (CH) and 109 liver cirrhosis (LC). ADAMTS13:act was measured by both the classic VWFM assay and the novel monoclonal antibody-based ELISA (Transfusion2006, 46:1444). The ADAMTS13:ag was quantified by a sandwich ELISA using two anti-ADAMTS13 murine monoclonal Abs (A10 and C7), and the UL-VWFM was analyzed by a SDS−0.9% agarose gel electrophoresis. Results: ADAMTS13:act in LC progressively decreased from the highest in Child A (mean 79%), to Child B (63%), to the lowest in Child C (30%), compared with CH (87%) and normal healthy subjects (N, 102%). The activity measured by VWFM assay highly correlated with that assayed by ELISA (r=0.75, p

Description: Introduction: Daily plasma exchange (PE) has become a definitive first-line treatment for acquired thrombotic thrombocytopenic purpura (TTP). However, it remains to be controversial whether platelet transfusion is harmful or not for patients with acquired TTP during initial treatment. Some articles showed that platelet transfusion was considered as hazardous because platelet transfusion might generate widespread fresh platelet aggregates in circulation, but others reported that the mortality rate was not different between patients with and without platelet transfusion. Herein, we conducted a retrospective analysis of a large cohort of patients with acquired idiopathic TTP (ai-TTP) in Japan evaluating whether platelet transfusion was associated with unfavorable outcomes. Patients and Methods: Our laboratory has been functioning as a nationwide referral center for thrombotic microangiopathies (TMAs) in Japan. We collected a large dataset of medical information on 1211 patients with TMA from March 2000 to December 2013. Among them, 263 were ai-TTP patients with severe deficiency (

Description: About 500 persons in Japan are estimated to have thrombotic thrombocytopenic purpura (TTP), which is a life-threatening disorder. In contrast with the USA and EU, off-label use of rituximab for TTP is not allowed by the Japanese public health insurance system. We therefore have limited experience of the effects of rituximab in Japanese patients with adult TTP and have not investigated possible ethnicity-related differences in its effects. To evaluate the safety and efficacy of rituximab in Japanese adult patients with TTP, we conducted an investigator-initiated phase 2 clinical trial in Japan between January and December of 2014. This study was sponsored by the Japanese government and the study design was based instructions from the regulatory agency, PMDA (Phamaceuticals and Medical Devices Agency). Adult Japanese patients with acquired TTP that had proven refractory to at least five plasma exchanges (PEX) or had serum concentrations of more than 2 BU/mL of ADAMTS13 inhibitor or both were eligible for this study. Rituximab was infused at 375 mg/m2 weekly for 4 weeks. The study protocol allowed use of both plasma exchange and corticosteroids with the investigational drug. Thirteen patients were registered and seven found to be eligible and treated with rituximab. All seven patients were evaluable for safety and six for efficacy. Three of these six (50%) were women; the median age was 41.5 years. Median platelet counts at baseline were 22 × 109/L. All of these six patients were refractory to previous PEX and ADMTS13 inhibitor concentrations were higher than 2 BU/mL in two (33%) of them. All six patients no longer required PEX after receiving rituximab treatment. Five (83%) and two (33%) patients achieved platelet counts of 100 × 109/L and 150 × 109/L 4 weeks after initiating rituximab, respectively. Median platelet counts at week 4 after commencing rituximab were 136 × 109/L (range: 64-201 × 109/L). The median time from initiating PEX to achieving platelet counts over 150 × 109/L was 22 days. By the study endpoint (4 weeks after initiating rituximab treatment), ADAMTS13 activity had normalized to 77.3% from 3.4%, accompanied by disappearance of ADAMTS13 inhibitor. Both anemia and neurological impairment also improved in all patients. The median percentages of peripheral blood CD20-positive B cells were 22.9% and 0.2% before and at the completion of treatment with rituximab, respectively. Serum IgG decreased slightly from 1,116 mg/dL at baseline to 921 mg/dL 4 weeks after rituximab treatment. Five of seven patients experienced adverse events, two of which were severe; namely septic shock and cytomegalovirus infection; no patients died. No unpublished severe adverse events were noted. No recurrence of TTP occurred during a median follow-up of 7 months. These data suggest that rituximab is safe and effective for acute refractory/high risk acquired TTP in Japanese patients and that there are no ethnic differences in effectiveness or safety of rituximab for adult TTP. However, there were too few patients to draw any firm conclusions. Further investigation is necessary to optimize the treatment schedule of rituximab. We are preparing to apply to the Japanese regulatory agency for approval of new indications for rituximab for TTP. Disclosures Off Label Use: Investigational drug, rituximab, was provided by Zenyaku Kogyo Co. Ltd.. Matsumoto:Baxter Bioscience: Consultancy.