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Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.

Introduction Immune-mediated thrombotic thrombocytopenic purpura (iTTP), or acquired TTP (aTTP), is a life-threatening thrombotic microangiopathy that requires prompt treatment to improve patient outcomes. Caplacizumab is a von Willebrand factor (VWF)-directed antibody fragment that rapidly inhibits VWF-platelet interaction and prevents microthrombi formation in iTTP. Based on efficacy and safety demonstrated in the Phase 3 HERCULES trial, caplacizumab, in conjunction with therapeutic plasma exchange (TPE) and immunosuppression, is approved in the USA and EU for aTTP. The aim of this Phase 2/3 study (NCT04074187), conducted in Japan, was to evaluate the efficacy and safety of caplacizumab in Japanese patients with iTTP. Methods Japanese patients aged ≥18 years with a clinical diagnosis of iTTP who had received ≤1 TPE were enrolled in this single-arm, open-label study. Patients received caplacizumab in conjunction with TPE and immunosuppression, during daily TPE, and for ≥30 days after discontinuation of TPE. Treatment extension was allowed for ≤8 weeks in case of persistent ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency defined by the investigator and patients were followed for 4 weeks after end of caplacizumab treatment. Primary endpoint was the proportion of patients with a recurrence of iTTP during the overall study period, assessed in the per-protocol (PP) population; recurrence rate ≤20% was the success criterion. Key secondary endpoints were assessed in the PP and modified intention-to-treat (mITT) populations. Treatment-emergent adverse events (TEAEs) were assessed in the safety population. PP population included patients who completed treatment and follow-up per protocol or had a recurrence of iTTP; mITT and safety populations included patients with ≥1 dose of caplacizumab. All analyses were descriptive. The study was conducted in accordance with the Declaration of Helsinki. Results A total of 21 patients were enrolled and treated with caplacizumab; 6 patients discontinued (adverse event: n=2, physician decision: n=4), and 15 patients were included in the PP population. In the mITT population, median age (range) was 59 (22-86) years; 16 (76%) patients presented with an initial episode; median (range) platelet count at baseline was 21.5 (8-78) ×10 9/L;10 (48%) patients received rituximab; median duration (range) of caplacizumab exposure during the overall treatment period was 35 (7-69) days. All patients in the PP population had ADAMTS13 activity Conclusions In Japanese patients with iTTP, caplacizumab in conjunction with TPE and immunosuppression was associated with a low rate of recurrence of iTTP and fast normalization of platelet count and organ damage markers. These findings are comparable to those in the HERCULES study, in which caplacizumab was associated with 12% recurrence rate and median (95% CI) time to platelet count normalization of 2.69 (1.89-2.83) days (Scully M, et al. N Engl J Med. 2019;380 [4]:335-346). Caplacizumab was well tolerated, and no new safety signals were identified in the Japanese population. Funding This research was funded by Sanofi. Figure 1 Figure 1. Disclosures Miyakawa: Sanofi: Research Funding; Zenyaku Kogyo: Consultancy; Sanofi: Consultancy; argenx: Consultancy, Research Funding. Imada: Celgene Co., Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.,: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co. Ltd.: Honoraria; Takeda Pharmaceutical Co. Ltd.: Honoraria; Novartis Pharma K.K.: Honoraria. Ichikawa: Sanofi: Honoraria; AstraZeneca: Honoraria; Chugai: Honoraria. Handa: Daiichi Sankyo: Research Funding; Janssen: Honoraria; BMS: Honoraria; Ono: Honoraria; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding; Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding. Matsushita: Shire/Takeda: Honoraria; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ/Sanofi: Honoraria; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational and investigational support; CSL Behring: Honoraria; JB: Honoraria; KMB: Honoraria; Nichiyaku: Honoraria; Octapharm: Honoraria; Sysmex: Honoraria; Baltaxa/Shire/Takeda: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: Educational and investigational support; Pfizer: Membership on an entity's Board of Directors or advisory committees; Kirin: Honoraria. Hashimoto: Sanofi KK: Current Employment, Other: May hold shares and/or stock options. Ohshima: Sanofi KK: Current Employment, Other: May hold shares and/or stock options. Tahara: Sanofi KK: Current Employment, Other: May hold shares and/or stock options. Tanaka: Sanofi KK: Current Employment, Other: May hold shares and/or stock options. Matsumoto: Sanofi: Consultancy; Takeda: Consultancy; Alexion Pharma: Consultancy; Asahi Kasei Pharma: Research Funding; Chugai Pharmaceutical: Research Funding; Alfesa Pharma: Patents & Royalties: ELISA for measuring ADAMTS13 activity.

Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib–von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13−/− and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13−/− mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13−/− compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a function-blocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWF-cleaving activity of ADAMTS13, provided visual evidence for a shear rate–dependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis.

A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.

To know the role and initiation mechanism of the interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF), we examined the effect of shear stress levels on platelet binding to vWF-coated plates using a cone-and-plate type viscometer capable of loading various levels of shear stress. The extent of platelet binding to immobilized vWF reached a plateau at the shortest period tested (20 seconds) under high shear stress (90 dyne/cm2), whereas 9 to 12 minutes was necessary for saturable platelet binding under static conditions. This shear effect, which was found to be dependent on the vWF-GP Ib interaction, was observed even under the lowest shear stress (1.5 dyne/cm2) examined. In contrast with the high shear effect previously reported to initiate the interaction of GP Ib with soluble vWF, these results indicate that relatively low levels of shear stress can promote the interaction of GP Ib with surface- immobilized vWF. This effect of shear stress was observed regardless of the manner in which vWF was immobilized, suggesting that immobilization itself and not, as previously hypothesized, a conformational change in vWF induced by direct adsorption to the surface is responsible for the enhanced GPIb binding. Thus, the present findings suggest that the vWF- GP Ib interaction contributes optimally to rapid platelet cohesion on a thrombogenic surface when vWF is in a static state and when platelets are moved by an appropriate rheological force such as low shear stress.

Hepatic ischemia-reperfusion (I/R) injury is a liver damage occurring during liver surgeries such as hepatic resection or transplantation, and denotes the major basis for graft dysfunction after transplantation. Although cellular and molecular mechanisms of hepatic I/R injury are complex and remain to be clarified, an excessive inflammatory response is assumed to play a role in this regard. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. In this context, we assumed that VWF may also be involved in the pathophysiology of hepatic I/R injury, in which VWF-dependent platelet activation or inflammatory responses could play a role at liver sinusoidal milieu. To test this hypothesis, we have used a mouse experimental model of hepatic I/R injury. Mice were anesthetized with sodium pentobarbital and a midline laparotomy was then performed on a heating pad. Blood supply for left lateral and median lobes of liver (approximately 70% of the liver mass) was interrupted by cross-clamping the hepatic artery and portal vein with a microvascular atraumatic clip for 90 min. Then a clip was taken off to provoke the reperfusion of hepatic blood flow, which was monitored on the surface of left lateral lobe by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The hepatic blood flow was measured again 24 h after reperfusion and mice were then sacrificed for blood collection and histological analysis of liver tissue. We compared 16 wild-type (WT) and 12 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 8-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. As compared to WT mice, restoration of hepatic blood flow was significantly greater in VWF-KO mice at 24 h after reperfusion (WT; 61± 17% vs. KO; 87 ± 17%, expressed as the percentage of pre-ischemic value). Consistent with the hepatic blood flow, the time-course analysis of serum alanine aminotransferase (ALT) at several time points after reperfusion revealed the lesser liver damages of KO mice (WT; 6898 ± 3270 and 1313 ± 621 IU/L vs. KO; 3043 ± 1320 and 478 ± 330 IU/L, at 3 h and 24 h after reperfusion, respectively). In addition, histological analysis confirmed that neutrophil infiltration in the liver tissue of KO mice was significantly reduced as compared to WT mice at 24 h after reperfusion. These impaired hepatic blood flow and ALT values as well as intensified neutrophil infiltration in WT mice were significantly improved to an extent comparable to those of KO mice by the bolus injection of recombinant human ADAMTS13 (3 μg/mouse equivalent to 2800 U/kg, n=12) just prior to the I/R operation. Our results altogether indicate that VWF-dependent inflammatory responses with neutrophil recruitment at ischemic sites are involved in pathophysiology of hepatic I/R injury, and functional regulation of VWF by ADAMTS 13 may serve as a promising therapeutic option for hepatic I/R injury. Disclosures No relevant conflicts of interest to declare.

Acute kidney injury (AKI), an abrupt loss of renal function, is often seen in clinical settings and its mortality remains high even in the developed countries. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. Recent mouse model studies demonstrated that VWF-mediated thrombotic and inflammatory responses could play a role in the disease progression of myocardial infarction or brain stroke. Thus, we assumed that VWF may also be involved in the pathophysiology of AKI, the major cause of which could be an insufficient renal circulation and/or inflammatory cell infiltration in the kidney. To test this hypothesis, we studied the relevant role of VWF in AKI in mouse model of acute ischemia-reperfusion (I/R) kidney injury. All mice used were male, 8-12 weeks of age, healthy and whose right kidney was surgically removed by the standard mouse nephrectomy procedure 1 week prior to the kidney I/R experiment. The preliminary experiments confirmed that surgical removal of mouse right kidney did not affect their general conditions including renal functions. Mice were anesthetized with inhaled isoflurane and then placed in an abdominal position on a heating pad. Surgical incision was given on the left side of back and the left kidney was brought out and kept outside during the operation. Both renal artery and vein were clamped at the renal hilus by a clamping clip for 30 min ischemia. Then a clip was taken off to provoke the reperfusion of renal blood flow, which was monitored by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The kidney was then put back in a body and skin incision was closed. The renal blood flow was measured again 30 h after reperfusion and mice were then sacrificed for blood collection. We compared 15 wild-type (WT) and 16 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME). Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. Although no difference was seen immediately after reperfusion, significantly (p < 0.05) higher renal blood flow at 30 h after reperfusion was confirmed in VWF-KO mice, as compared to WT (KO; 24.0±2.3 vs. WT; 15.1±1.46 ml/min/100g of kidney weight, and the reperfusion/base flow ratio: KO; 1.0±0.07 vs. WT; 0.6 ±0.07). Consistent with the renal blood flow data, the serum creatinine value at 30 h after reperfusion were significantly (p < 0.05) lower in VWF-KO mice than WT (KO; 2.77±0.11 vs. WT; 3.15±0.11 mg/dl). Our results suggest that VWF does play a role in the pathogenesis of AKI, in which VWF-dependent thrombotic or inflammatory responses may trigger thrombotic ischemia or endothelial damages of vascular bed in the kidney. Thus, proper functional regulation of VWF would be beneficial for better microcirculation and vessel functions in the kidney, suggesting a novel therapeutic potential against AKI. Disclosures No relevant conflicts of interest to declare.

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.

Sepsis is a serious inflammatory response syndrome, in which systemic activation of both inflammatory and coagulation pathways are provoked by severe microbial infection. In addition to the known hemostatic functions, von Willebrand factor (VWF) is recently assumed to participate in a cross-talk between inflammation and thrombosis. Indeed, recent mouse model studies demonstrated that proper functional regulation of VWF-dependent inflammatory responses by ADAMTS13 considerably improved the disease progression of brain stroke or myocardial infarction. However, little is known about the detailed mechanisms or relevant role of VWF functions in inflammation. We therefore studied the physiologic relevance of VWF-dependent inflammatory responses in a mouse model of experimental sepsis by cecal ligation and puncture (CLP). The mouse CLP was performed according to the established standard protocol (Rittirsch D, et al., Nat Protoc, 2009). Briefly, mouse cecum was ligated distal to the ileocecal valve under laparotomy, punctured with 18 gauge needle and gently pressed until a small drop of stool appeared. The cecum was returned to the peritoneal cavity and 200 μL of saline was injected into the cavity to avoid dehydration before body wall and skin incision were closed with a 4-0 Sofsilk. We compared 17 wild-type (WT) and 17 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 10-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all (WT or KO) mice during the CLP operation. Kaplan-Meier analysis revealed the significantly (p < 0.05) lower survival rate of KO mice than that of WT (KO; 23.5% vs. WT; 58.8% at the Day 7 of CLP). The impaired survival rate of KO mice was restored by the bolus administration of human VWF (n=21, 2.5 U/mouse) to an extent comparable to that of WT. Peripheral blood analysis of KO mice at 24 hours after CLP showed the severe leukocytopenia with sharp decrease of neutrophils in blood, as compared to WT. In addition, formation of walled-off abscess was confirmed at the peri-cecal space in all the WT and KO mice alive even at the Day 7 of CLP, while such focal abscess formation was not found in mice died before the Day 3 of CLP. Thus, our results altogether indicate that VWF could play a role on the recruitment or accumulation of neutrophils for microbial killing at the local inflammatory focus. VWF-mediated platelet aggregate formation in peripheral capillary vessels then could shut down the local microcirculation, thereby blocking systemic microbial expansion as a crucial biological defense mechanism. Disclosures Shima: Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Mural thrombus formation at sites of damaged vessel wall, essential for both physiologic hemostasis and pathological thrombosis, is established by platelet adhesion/aggregation and blood coagulation mechanisms. Although tissue factor (TF) is up-regulated upon vessel wall damage and plays a pivotal role in the latter process, its functional relevance under physiologic blood flow conditions is poorly understood. Using an in vitro perfusion chamber system, we have therefore studied the relevant role of TF in thrombus formation mediated by von Willebrand factor (VWF), a distinctive flow-dependent thrombogenic surface, under whole blood flow conditions with varying shear rates. Human recombinant TF (Innobin) were co-coated with purified VWF (100 ug/ml) onto a glass plate to prepare ‘surface-immobilized TF/VWF complex’. Surface density of immobilized TF, evaluated by the ELISA-based assay using an anti-TF monoclonal antibody, was increased in a concentration-dependent and saturated manner by soluble TF (1-100 pM) added on a plate. Citrated whole blood, recalcified with 8 mM CaCl2 prior to perfusion, was perfused over a VWF-surface in the presence or absence of surface-immobilized TF. Platelet adhesion and aggregation was evaluated by the surface coverage of generated thrombi in a defined area after 5-min perfusion. Mural thrombi formed on VWF-surface were also double-stained with fluorescently labeled anti-fibrin and anti-fibrinogen antibodies. Fibrin generation was evaluated by confocal laser scanning microscopy as a ratio of fibrin relative to fibrinogen fluorescence within mural thrombi. As a result, surface-immobilized TF significantly augmented flow-dependent fibrin generation as a function of increasing surface density of TF under both low (250 s-1) and high (1500 s-1) shear rate conditions. In this regard, soluble TF, when added to sample blood, similarly increased intra-thrombus fibrin generation in a dose-dependent manner in the absence of immobilized TF. However, coagula formation in sample blood was enormously amplified by soluble TF during perfusion, as judged by the flow-path occlusion time. In addition to the enhancing effects on fibrin generation, immobilized TF significantly up-regulated VWF-dependent platelet adhesion and aggregation under high shear rate conditions, albeit with no appreciable effects under low shear rate conditions. These results suggest a synergistic functional link between immobilized TF and VWF in mural thrombus formation under high shear rate conditions. Our results clearly illustrate the thrombogenic potentials of two distinct forms (soluble or surface-immobilized) of TF, in which surface-immobilized TF plays a concerted role on VWF-dependent thrombus formation with lesser risk of systemic hypercoagulability which may be induced by circulating soluble TF under high shear rate conditions. Disclosures: No relevant conflicts of interest to declare.

Abstract 1077 The adhesive protein von Willebrand factor (VWF) plays an essential role on physiologic hemostasis, mediating platelet aggregation under high shear stress conditions. However excessive functions of VWF could cause thrombotic occlusion of microvasculature such as arterial capillaries, where blood flow creates a typical high shear stress. The VWF-cleaving protease ADAMTS13 is therefore thought to down-regulate precisely the VWF function to maintain enriched microcirculation. In this context, we hypothesized that this ADAMTS13 role might contribute to better donor cell homing and engraftment in various cell therapy approaches, in which fluent blood flow could be critical in the microcirculation system. To test this hypothesis, we investigated the essential role of ADAMTS13 on the donor cell engraftment using bone marrow transplantation (BMT) model in Adamts13 −/− and wild-type mice. Irradiated recipient mice were received 2 × 106 GFP positive cells from the sex-matched GFP donor mice. All of irradiated recipient mice without receiving BMT died within 21 days. Although there is no difference between Adamts13 −/− and wild-type mice in survival rate after 7days of BMT, Kaplan-Meier analysis revealed that the percent ratio of survival rate starts significantly declining after 14 days of BMT in the group of Adamts13 −/− mice. The successful cell engraftment in BMT was assessed by the number of GFP-positive neutrophils in peripheral blood at the several time points from BMT. As a result, the duration achieving the number of GFP-positive neutrophils over 500/μL was found to significantly delay in the Adamts13 −/− mice, as compared with the wild-type mice (20.2±3.8 days vs. 14.4±3.3 days). However, histological examination during the whole observation periods could not detect any typical thrombotic lesions of micro vessels developed in both wild-type and Adamts13 −/− mice. In addition, the delayed cell engraftment observed in the Adamts13 −/− mice became normalized by the bolus administration of recombinant ADAMTS13 (10 μg/mouse) at the day 0 of BMT. Bone marrow analysis at the day 1 of BMT revealed that the number of GFP-positive blood cells in bone marrow was significantly reduced in the Adamts13 −/− mice as compared with the wild-type mice, which could result in the delayed cell expansion at the day 7 and day14 of BMT in Adamts13 −/− mice. The single bolus injection of recombinant ADAMTS13 was found to fully correct the delayed cell expansion in bone marrow in the Adamts13 −/− mice. Our results indicate that the regulation of VWF-mediated thrombotic or inflammatory responses by ADAMTS13 could contribute to better microcirculation which could be critical for efficient donor cell homing and engraftment in BMT, suggesting a therapeutic potential of ADAMTS13 in cell therapy approaches. Disclosures: Soejima: The Chemo-Sero-Therapeutic Research Institute: Employment.

Abstract 2175 The metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers, controlling excessive VWF functions and preventing thrombotic occlusion of microvasculature. We previously reported that ADAMTS13 deficiency aggravated the extent of brain ischemic stroke in a mouse model of middle cerebral arterial occlusion, suggesting the relevant role of ADAMTS13 in the pathophysiology of brain stroke (Fujioka, et al. Blood, 2010; 115: 1650). These results raised the possibility that the functional regulation of VWF by ADAMTS13 could also play a role in coronary ischemic events such as myocardial infarction. To address this issue, we have used a mouse model of experimental myocardial infarction. The left anterior descending coronary artery in mice was ligated at 2 mm downstream from the origin under thoracotomy with ventilator-assisted respiration, and the cardiac function was evaluated with M-mode echocardiography after 7 days of operation. We compared 20 wild-type (WT) mice and 20 Adamts13 −/− (KO) mice, all of which were 12–14 weeks of age, healthy and fertile. Significantly (p < 0.01) decreased ejection fraction (EF; 44.0±6.7%) and increased left ventricular end-diastolic diameter (LVDd; 4.68±0.69 mm) of KO mice, as compared to WT (EF; 62.7±13.0% and LVDd; 3.77±0.56 mm, respectively), revealed that cardiac functions were apparently more impaired in KO mice. In addition, these reduced cardiac functions observed in KO mice were improved to an extent comparable to those of WT mice by the bolus injection of recombinant human ADAMTS13 (rhADAM; 3 μg/mouse, n=20) just after the operation (KO mice + rhADAM, EF; 58.2.±9.9% and LVDd; 3.16±0.52 mm). Consistent with echocardiography data, histological studies demonstrated the significantly (p < 0.01) higher infarct ratio in myocardium of KO mice (WT; 37.3±18.4%, KO; 59.1±16.3%, KO + rhADAM; 33.7±24.4%). Our results indicate that ADAMTS13, as seen in the case of brain ischemic stroke, plays a protective role for myocardium in coronary artery ischemia, improving myocardial functions and the severity of heart failure. Proper functional regulation of VWF-dependent thrombotic or inflammatory responses by ADAMTS13 could reduce thrombotic occlusion of microvasculature including leukocyte plugging, contributing to better local microcirculation which is crucial for tissue or organ functions. Disclosures: No relevant conflicts of interest to declare.

Abstract 3349 Background: Sepsis is a critical consequence of self defense to infection, which includes inflammatory and hypercoagulant response. Many stimulatory mediators involved in these responses cause the Weibel-Palade body exocytosis through endothelial activation, leading to surface expression of P-selectin and ultra-large von Willebrand factor (ULVWF), and simultaneously release of VWF-propeptide on a equimolar basis to ULVWF. Where ADAMTS13 is functionally inactivated, ULVWF would not be proteolysed and cause platelet thrombosis in the microcirculation, leading to organ failure. Therefore, the balance of the ULVWF and ADAMTS13 may indicate the prothrombotic state of the patients. However, it is difficult to estimate the amount of ULVWF expressed. It is reasonable to think that plasma VWF propeptide levels will reflect more precisely the extent of expressed ULVWF and P-selectin rather than VWF level. These allow us to speculate that the balance of VWF propeptide and ADAMTS13 can be associated with magnitude of prothrombotic and inflammatory response. Aim: To investigate whether the levels of molecules mentioned above are associated with the disease severity of sepsis or inflammation during the clinical course. Method: Inclusion criteria are as follows, 1) severe sepsis or septic shock defined by the Society of Critical Care Medicine Consensus Conference Committee 2) Platelets count less than 120,000/μL. SOFA(sequential organ failure assessment)score, APACHEII score, Creatinine, VWF propeptide, VWF, P-selectin, ADAMTS13, TNF-alfa, IL-6 were measured at day 0, 3, 5, 7 (day 0 is the day when the diagnosis as severe sepsis or septic shock was made), and each other's correlation was studied. Result and Discussion: VWF propeptide level was increased to 293.8+/−153.8% at day 0, and gradually decreased day by day. VWF was increased to 212.3+/− and slightly increased during the course. P-selectin was not increased during the course. The plasma level of ADAMTS13 at day 0 was decreased to 25+/− 8.7%, and increased during the course in the survivors, however almost not changed in the non-survivors. As we speculated, the value of VWF propeptide/ ADAMTS13 were well correlated to SOFA score, Creatinine, APACHEII, TNF-alfa almost on each study day. Conclusion: This result shows that the ratio of VWF propeptide to ADAMTS13 can be used to estimate disease severity of sepsis and magnitude of inflammation. This will improve our understanding of underlying pathophysiology of sepsis. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. 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.

Abstract 2293 Background: Recently, a recombinant human soluble thrombomodulin (rTM) has become commercially available for patients with disseminated intravascular coagulation (DIC) in Japan, which is composed of the active, extracellular domain of thrombomodulin. However, its anti-thromobotic and anti-inflammatory effect during the clinical course in patients with DIC has not been reported. Aim: To investigate the effect of rTM on mortality, haemostatic disturbance, and inflammation in septic patients with DIC. Methods: The patients with sepsis who met the Japanese diagnostic criteria for acute DIC and showed a level of antithrombin (AT) lower than 70% were recruited and divided into two groups, one group (Control group) treated with AT products and Gabexate mesilate (GM), and the other group (TM group) treated with rTM (0.06 mg kg(-1) for 30 min, once daily) in addition to AT and GM. The effect of rTM during the clinical course were investigated from the differences between TM and Control groups in mortality, DIC scoring, haemostatic and inflammatory markers on days 0, 3, 5, 7 (day 0 is just before initiation of therapy). Results & Discussion: Eighteen patients were included in the TM group, and 16 patients in the Control group. There were no differences in APACHEII score at day 0, DIC score at day 0, or mortality at day 28 between the groups. The effects of the rTM were as follows; 1) Patients in the TM group showed earlier DIC resolution at day 5 than Control at day 7. 2) Hypercoagulable state expressed by the increased levels of soluble fibrin monomer (SF) or thrombin-antithrombin complex (TAT) was improved more quickly in TM group compared with Control group, suggesting that rTM decreased thrombin generation. 3) AT was increased much more at day 3 after AT product administration in TM group than in Control group, which also can be explained by suppression of thrombin generation by rTM. 4) Increased levels of the complex of α2PI/plasmin, D-dimer, and fibrin/fibrinogen degradation product were also improved earlier in TM group than Control group. 5) Plasmin-alfa2 plasmin inhibitor complex (PIC) was significantly lower at day 7 in TM group than Control group, suggesting anti-fibrinolytic effect of rTM. 6) Decreased level of ADAMTS13 increased more quickly in TM group. 5) TNF-α, IL-6, HMGB1 were decreased significantly at day 3 compared with day 1 only in TM group. These anti-inflammatory effect can be evoked through direct sequestration of HMGB1 by rTM or decreased thrombin generation by rTM, because thrombin is a strong pro-inflammatory molecule through PAR1. Conclusion: rTM may be beneficial in improving septic DIC via its anti-thrombotic and anti-inflammatory properties. Disclosures: No relevant conflicts of interest to declare.

Background: Severe acute pancreatitis (SAP) frequently progresses to pancreatitis-associated multiorgan failure (MOF) with high mortality. Decreased plasma ADAMTS13 activity (ADAMTS13:AC) results in the accumulation of unusually large von Willebrand factor multimers (UL-VWFM) and the formation of platelet thrombi, ultimately leading to MOF. We demonstrated that the imbalance between decreased ADAMTS13:AC and increased UL-VWFM could contribute to SAP pathogenesis through enhanced thrombogenesis, and serve as an early prognostic indicator for SAP patients (Scand J Gastroenterol, 2008, 26:1). Endotoxin has been considered to be the principle activator of the systemic inflammatory response syndrome, which predisposes patients for MOF and/or pancreatic necrosis, ultimately leading to SAP. We investigated the relationship of endotoxin to ADAMTS13:AC and its related parameters, and tried to explore their potential role on the development of MOF in patients with SAP. Methods: We sequentially determined plasma endotoxin concentration, ADAMTS13:AC and its related parameters in 13 SAP patients (APACHE-II score mean 6.6 ± 2.7), who were admitted into intensive care unit of our hospital between 2004 and 2006. Eleven patients were survivors and two were non-survivors whose APACHE II scores were 10 and 12 died of MOF, respectively. The degree of MOF was evaluated according to the SOFA score. Endotoxin concentration was determined by a chromogenic substrate assay (Toxicolor LS –M Set, Seikagaku Kogyo Co.) with kinetic analysis after pretreatment with detergent, Triton X-100, and heating at 70 °C for 10 min. Plasma ADAMTS13:AC was determined by a sensitive chromogenic ELISA (ADAMTS13-act-ELISA: Kainos Inc.). Plasma UL-VWFM was analyzed by a vertical SDS-1.0% agarose gel electrophoresis. Plasma VWF antigen (VWF:AG), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor -α (TNF-α) were measured by ELISA. Results: In normal healthy controls (n=20), plasma endotoxin concentration was 7.9±1.7 pg/ml (mean ± SD). The concentration in the SAP patients significantly increased at day 1 (means 65 pg/ml, p Conclusion. The imbalance between decreased ADAMTS13:AC and increased UL-VWFM is closely related to enhanced endotoxemia, which may contribute to the development of SAP and subsequent MOF through enhanced thrombogenesis.

Background: The proteolytic activity of human ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers, controlling excessive platelet aggregation and preventing microvascular thrombus formation. Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP) and patients with TTP often have neurological deficits such as stupor or coma. Therefore, ADAMTS13 appears necessary for vascular homeostasis in the brain and may also influence the response to brain injury during ischemic stroke. Method and Result: We investigated the role of ADAMTS13 in a mouse middle cerebral arterial occlusion (MCAO) model of ischemia-reperfusion injury in the brain. We compared 24 wild type mice (WT) and 24 ADAMTS13 gene deleted mice (KO), which are healthy and fertile. All mice were males 6–8 weeks of age. Investigators were blinded to the genotype until all analyses were finished. We applied MCAO for 30 minutes followed by 23.5 hours of reperfusion. The cerebral blood flow (CBF) around the cortex of the ischemic region was measured by laser Doppler flowmetry for 60 minutes after MCAO. In both WT and KO mice, the CBF decreased to less than 20% of baseline during MCAO and returned to normal immediately after reperfusion. However, during the subsequent 30 min the CBF decreased to 34.6±5.8% of baseline for KO mice compared to 83.2±6.8% of baseline for WT mice (P < 0.01) and remained significantly decreased at 24 hours, suggesting that ADAMTS13 deficiency promotes thrombosis after reperfusion injury. The infarction volumes of the brain were determined from the area not stained by 2,3,5,-triphenyltetrazolium chloride (TTC) 24 hours after MCAO. KO mice had significantly increased volume of brain infarction compared with WT (31.0±6.5mm3 vs 11.4±1.9 mm3, P < 0.01). The degree of post-ischemic inflammation was estimated by Western blotting of plasma HMGB1 (high-mobility group box1) 24 hours after MCAO. Plasma HMGB1 was increased to 34.4 ± 5.3 ng/ml in KO mice, compared to 19.6 ± 3.5 ng/ml in WT mice (P < 0.05). The KO and WT mice did not differ during the MCAO procedure in body temperature, survival (80% vs 85%) at 24 hours, prothrombin time, arterial pH, pO2 or pCO2. Conclusion: ADAMTS13 deficiency aggravates the extent of persistent brain ischemia, infarct volume and inflammatory response after brief MCAO. Therefore, ADAMTS13 plays a neuroprotective role against ischemia-reperfusion injury.

Background: A severe form of acute pancreatitis, severe acute pancreatitis (SAP), frequently develops pancreatitis-associated multiorgan failure (MOF) followed by systemic microcirculatory disturbance with a high mortality. ADAMTS13 has been focused on the occurrence of thrombotic thrombocytopenic purpura (TTP). TTP has been reported to cause acute pancreatitis in a few percents, and recent study indicates that some acute pancreatitis may be a triggering event for TTP. We sequentially determined plasma ADAMTS13 activity (ADAMTS13:AC) and its related parameters in patients with SAP, and thereby, tried to explore their potential role on the development of MOF. Methods: Subjects studied were 13 patients with SAP, who were admitted into the department of emergency and critical care medicine of our hospital. The etiology was alcohol in 7, idiopathic in 3, common bile duct stones in 2, and post endoscopic retrograde cholangiopancreatography in 1. Eleven patients were survivors and two were non-survivors. Two of 4 patients with MOF were non-survivors. The severity was scored according to APACHE-II system. ADAMTS13:AC was determined using a commercially available ADAMTS13-act-ELISA (Kainos Inc., Tokyo). Plasma levels of VWF antigen (VWF:AG), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis-α (TNF-α) were measured by ELISA. Unusually large VWF multimer (UL-VWFM) was analyzed by a vertical SDS -1.0% agarose gel electrophoresis system. Results: ADAMTS13:AC significantly decreased at day 1 (mean 39%, p Conclusion: Markedly decreased ADAMTS13:AC together with increased amounts of UL-VWFM was closely related to the severity of pancreatitis, an intense systemic inflammatory response, and prognosis in patients with SAP. These results indicate that the imbalance between the enzyme and its substrate may involve in the development of acute pancreatitis and subsequent MOF through enhanced thrombogenesis.