Unusually Large Von Willebrand Factor Multimers Research Articles

COVID-19 microthrombosis: unusually large VWF multimers are a platform for activation of the alternative complement pathway under cytokine storm.

ADAMTS13, a metalloproteinase, specifically cleaves unusually large multimers of von Willebrand factor (VWF), newly released from vascular endothelial cells. The ratio of ADAMTS13 activity to VWF antigen (ADAMTS13/VWF) and indicators of the alternative complement pathway (C3a and sC5b-9) are both related to the severity of COVID-19. The ADAMTS13/VWF ratio is generally moderately decreased (0.18–0.35) in patients with severe COVID-19. When these patients experience cytokine storms, both interleukin-8 and TNFα stimulate VWF release from vascular endothelial cells, while interleukin-6 inhibits both production of ADAMTS13 and its interaction with VWF, resulting in localized severe deficiency of ADAMTS13 activity. Platelet factor 4 and thrombospondin-1, both released upon platelet activation, bind to the VWF-A2 domain and enhance the blockade of ADAMTS13 function. Thus, the released unusually-large VWF multimers remain associated with the vascular endothelial cell surface, via anchoring with syndecan-1 in the glycocalyx. Unfolding of the VWF-A2 domain, which has high sequence homology with complement factor B, allows the domain to bind to activated complement C3b, providing a platform for complement activation of the alternative pathway. The resultant C3a and C5a generate tissue factor-rich neutrophil extracellular traps (NETs), which induce the mixed immunothrombosis, fibrin clots and platelet aggregates typically seen in patients with severe COVID-19.

Analysis focusing on plasma von Willebrand factor in pachychoroid neovasculopathy and age-related macular degeneration

Pachychoroid neovasculopathy (PNV) is a new concept of macular disorder. Some cases diagnosed as age-related macular degeneration (AMD) have been re-diagnosed as PNV. However, the biological features of PNV are still uncertain. The purpose of this study was to compare PNV and AMD by analyses focusing on von Willebrand factor (VWF) and complement factor H (CFH). Ninety-seven patients who were previously diagnosed with treatment naïve AMD were enrolled in this study. They were re-classified as either PNV or AMD based on the clinical criteria and 33 patients were classified as PNV and 64 patients as AMD. We examined the clinical data, analyzed VWF multimer and two genetic polymorphisms (I62V and Y402H) in the CFH. PNV group was significantly younger than AMD group (P = 0.001). In both I62V and Y402H, there were no significant differences between PNV and AMD while the recessive homozygous (AA) was found only in PNV group in I62V. The presence of unusually large VWF multimers (UL-VWFMs) and subretinal hemorrhages were significantly higher in PNV than in AMD (P = 0.045, P = 0.020, respectively). Thus, the residual UL-VWFMs may result in platelet thrombosis and hemorrhages in the choriocapillaris of PNV. In conclusion, our results suggest the biological differences between PNV and AMD.

Ischemia-Reperfusion Injury with Pringle's Maneuver Induces Increasing of Unusually Large Von Willebrand Factor Multimers after Hepatectomy

Background/Aims: Von Willebrand factor (VWF) is exclusively synthesized in vascular endothelial cells and released into the plasma as unusually large VWF multimers (UL-VWFM). ADAMTS13 is a metalloproteinase produced primarily in hepatic stellate cells and specifically cleaves the VWF. Since UL-VWFM have a strong activity of platelet aggregation, increase of UL-VWFM leads to platelet clumping and/or thrombus formation, resulting in microcirculatory disturbance. On the other hand, ischemia-reperfusion injury of the liver is an imperative event in liver surgery due to compression of the liver and/or Pringle's maneuver. Many previous studies have indicated that vascular endothelial cell is a major target of ischemia-reperfusion injury of the liver. Thus, we hypothesized UL-VWFM increases markedly by stimulating vascular endothelial cells with ischemia-reperfusion injury. However, little information is available regarding the alterations in VWF, ADAMTS13 and especially in UL-VWFM after hepatectomy. The aim of this study is to clarify the effect of hepatectomy on UL-VWFM.Methods: A total 35 patients who underwent a hepatectomy were eligible for the study. Blood samples were obtained for analysis at the following time points: preoperation, intraoperative period, and on the first, second, third, fifth, seventh postoperative days. Plasma levels of ADAMTS13 activity and VWF antigen were measured by the ELISA method. Multimer analysis of plasma VWF was performed according to the method of Ruggeri and Zimmerman. High molecular weight bands that were not detected in normal plasma were defined as UL-VWFM. UL-VWFM was quantified from images of VWF multimer analysis by NIH image. We calculated the UL-VWFM index, proposed in this study, which was defined as the ratio of the UL-VWFM area of a patient to that of normal plasma. All UL-VWFM indexes of 40 healthy volunteers were less than 1.00%. Therefore, UL-VWFM positive was defined as a UL-VWFM index of more than 1.00%. The patients were categorized and analyzed with respect to the positive or negative of UL-VWFM after hepatectomy. The values were expressed as median (range).Results: Plasma levels of ADAMTS13 activity were significantly decreased from 61.0% (27.7-126.2%) in preoperation to 37.4% (20.2-71.4%) in postoperative day 7 after hepatectomy (p <0.001). Plasma levels of VWF antigen were significantly increased from 172.1% (80.5-412.8%) in preoperation to 361.0% (154.7-745.8%) in postoperative day 2 after hepatectomy and remained continuously high until postoperative day 7 (p <0.001). The intraoperative plasma levels of ADAMTS13 activity, VWF antigen were not significantly altered. The UL-VWFM index were significantly increased from 0.2% (0.0-7.8%) in preoperation to 4.2% (0.1-16.3%) in postoperative day 7 after hepatectomy (p <0.001). The patients were divided into 2 groups with respect to the positive or negative of UL-VWFM after operation. Seven patients remained UL-VWFM negative after operation. UL-VWFM index were changed from negative in preoperation to positive after operation in 22 patients. Preoperative characteristics including ADAMTS13 activity, VWF antigen levels and liver functional reserve were similar in the two groups of UL-VWFM positive and negative. In surgery-related factors, the time of duration of Pringle's maneuver in UL-VWFM positive group were longer than those in UL-VWFM negative group (p=0.001). In addition, the volume of blood loss in UL-VWFM positive group was greater than those in UL-VWFM negative group (p=0.003). Multivariable analysis revealed that the time of duration of Pringle's maneuver [OR, 1.049 95%CI, 1.001-1.098; p=0.043] was significantly associated with increasing UL-VWFM after hepatectomy. Moreover, UL-VWFM index was significantly correlated with time of duration of Pringle's maneuver (r=0.444, p=0.017).Conclusion: In this study, we demonstrated that plasma UL-VWFM increases after hepatectomy due to ischemia-reperfusion injury with Pringle's maneuver. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

COVID-19 Sepsis: Pathogenesis and Endothelial Molecular Mechanisms Based on "Two-Path Unifying Theory" of Hemostasis and Endotheliopathy-Associated Vascular Microthrombotic Disease, and Proposed Therapeutic Approach with Antimicrothrombotic Therapy.

COVID-19 sepsis is characterized by acute respiratory distress syndrome (ARDS) as a consequence of pulmonary tropism of the virus and endothelial heterogeneity of the host. ARDS is a phenotype among patients with multiorgan dysfunction syndrome (MODS) due to disseminated vascular microthrombotic disease (VMTD). In response to the viral septicemia, the host activates the complement system which produces terminal complement complex C5b-9 to neutralize pathogen. C5b-9 causes pore formation on the membrane of host endothelial cells (ECs) if CD59 is underexpressed. Also, viral S protein attraction to endothelial ACE2 receptor damages ECs. Both affect ECs and provoke endotheliopathy. Disseminated endotheliopathy activates two molecular pathways: inflammatory and microthrombotic. The former releases inflammatory cytokines from ECs, which lead to inflammation. The latter initiates endothelial exocytosis of unusually large von Willebrand factor (ULVWF) multimers and FVIII from Weibel–Palade bodies. If ADAMTS13 is insufficient, ULVWF multimers activate intravascular hemostasis of ULVWF path. In activated ULVWF path, ULVWF multimers anchored to damaged endothelial cells recruit circulating platelets and trigger microthrombogenesis. This process produces “microthrombi strings” composed of platelet-ULVWF complexes, leading to endotheliopathy-associated VMTD (EA-VMTD). In COVID-19, microthrombosis initially affects the lungs per tropism causing ARDS, but EA-VMTD may orchestrate more complex clinical phenotypes, including thrombotic thrombocytopenic purpura (TTP)-like syndrome, hepatic coagulopathy, MODS and combined micro-macrothrombotic syndrome. In this pandemic, ARDS and pulmonary thromboembolism (PTE) have often coexisted. The analysis based on two hemostatic theories supports ARDS caused by activated ULVWF path is EA-VMTD and PTE caused by activated ULVWF and TF paths is macrothrombosis. The thrombotic disorder of COVID-19 sepsis is consistent with the notion that ARDS is virus-induced disseminated EA-VMTD and PTE is in-hospital vascular injury-related macrothrombosis which is not directly related to viral pathogenesis. The pathogenesis-based therapeutic approach is discussed for the treatment of EA-VMTD with antimicrothrombotic regimen and the potential need of anticoagulation therapy for coinciding macrothrombosis in comprehensive COVID-19 care.

Sepsis Produces Microthrombosis and Not Coagulation: Implications for Sepsis Therapy

Abstract The hemostatic abnormalities which may accompany sepsis have been attributed to disseminated intravascular coagulation (DIC) initiated by tissue factor (TF) activating FVII leading to TF-FVII a complex formation and triggering the extrinsic coagulation cascade. Recently, a new theory of hemostasis has been proposed in which sepsis is associated with endothelial damage releasing unusually large von Willebrand factor (ULVWF). The ULVWF multimers, following exocytosis from sepsis-induced endothelialopathy, become anchored to the endothelial cell membrane as elongated strings which recruit platelets from the circulation forming platelet-ULVWF complexes which become microthrombi. The process is called microthrombogenesis and the resultant disorder is disseminated intravascular thrombosis (DIT) which is a major factor leading to organ dysfunction. Thus, the sepsis-associated coagulopathy is not a coagulation disorder, but is a microthrombotic disorder. This conceptual reinterpretation entails a significant paradigm shift in the understanding of sepsis pathophysiology and treatment.

Link between von Willebrand factor multimers, relapses and coronary microcirculation in patients with thrombotic thrombocytopenic purpura in remission

IntroductionADAMTS13 deficiency results in unusually large von Willebrand factor (ULVWF) multimers in the circulation and a higher risk of microthrombi due to high shear stress. In patients treated for acquired thrombotic thrombocytopenic purpura (TTP), a persistently severe ADAMTS13 deficiency (<10%) in remission is associated with more relapses. A reduced plasma ADAMTS13 activity and increased VWF levels are associated with a higher risk of myocardial infarction. Assessing coronary flow reserve (CFR) enables a better cardiovascular risk stratification: a lower CFR correlates inversely with cardiovascular risk. The aim of the study was to establish whether patients with TTP in remission have an impaired coronary microcirculation, in terms of a lower CFR, and whether there is any correlation between ADAMTS13 activity, the presence of ULVWF multimers, and the occurrence of relapses. MethodsThe clinical information and hemostatic parameters of 24 patients with TTP in remission managed at our center were analyzed. The CFR was assessed in a subgroup of the TTP patients and compared with a control group consisting of 50 healthy volunteers. ResultsThe CFR was statistically lower in patients in remission of TTP than in controls, but there were no differences between TTP patients with normal and lower CFR. The occurrence of relapses correlated with the presence of ULVWF multimers and with a residual ADAMTS13 activity. ConclusionsWhen compared with healthy controls, TTP patients in remission have an impaired coronary microcirculation and the occurrence of relapses in the former reveal the presence of ULVWF multimers.

Intravitreal injection of aflibercept, an anti-VEGF antagonist, down-regulates plasma von Willebrand factor in patients with age-related macular degeneration

We investigated the association between von Willebrand factor (VWF) and exudative age-related macular degeneration (AMD) in 114 Japanese patients. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitor is the most effective therapy for AMD. Therefore, we analyzed changes of VWF antigen (VWF:Ag) and VWF multimers (VWFMs) after intravitreal injection of aflibercept, an anti-VEGF antagonist. The relationship between polymorphisms in complement factor H (p.Y402H and p.I62V) and AMD was previously reported. In our patients, p.I62V, but not p.Y402H, was significantly associated with an increased risk of AMD. Pre-treatment plasma levels of VWF:Ag in patients with AMD were significantly higher than those in controls. Unusually large VWFMs (UL-VWFMs) were detected in the majority of AMD patients with concurrent vitreous or subretinal hemorrhage. After intravitreal injection of aflibercept, plasma levels of VWF:Ag and VEGF-A were significantly decreased. UL-VWFMs disappeared after aflibercept injection in three cases, but persisted even 1 month after injection in the other five cases. In conclusion, plasma VWF:Ag levels were significantly elevated in patients with AMD, and decreased after intravitreal aflibercept injection. VWF may play an important role in the pathophysiology of AMD, and aflibercept might improve AMD by reducing plasma levels of VWF in addition to VEGF-A.

Decreased activity of plasma ADAMTS13 are related to enhanced cytokinemia and endotoxemia in patients with acute liver failure.

Deficient ADAM metalloproteinase with thrombospondin type-1 motif, member 13 (ADAMTS13) activity (ADAMTS13:AC) results in the accumulation of unusually large von Willebrand factor multimers (UL-VWFM) and causes microcirculatory disturbances and multiple organ failure, while endotoxins trigger the activation of a coagulation cascade. The objective of the present study was to explore the role of ADAMTS13 in endotoxemia in patients with acute liver failure (ALF). Plasma concentrations of endotoxin and cytokines, including interleukin (IL)-6 and IL-8, and activity of the plasma ADAMTS13 inhibitor were determined, along with ADAMTS13:AC, the VWF antigen (VWF:Ag) and UL-VWFM, in 27 patients with acute hepatitis (AH), 11 patients with ALF, and 10 healthy controls. IL-6 and IL-8 concentrations on admission were significantly higher in patients with ALF than in those with AH or in healthy controls. ADAMTS13:AC concomitantly decreased and VWF:Ag progressively increased with increasing cytokine concentrations from the normal range to >100 pg/ml. The inhibitor was detected in 8 patients with ALF (0.6 to 2.4 BU/ml) and 6 patients with AH (0.6 to 0.8 BU/ml). Patients with the inhibitor reported lower ADAMTS13:AC, higher VWF:Ag and lower functional liver capacity than those without the inhibitor. Collectively, the findings suggested that decreased ADAMTS13:AC and increased VWF:Ag may be induced by pro-inflammatory cytokinemia as well as the presence of the ADAMTS13 inhibitor, both of which may be closely related to enhanced endotoxemia in patients with ALF.

Von Willebrand Factor Deposition and ADAMTS-13 Consumption in Allograft Tissue of Thrombotic Microangiopathy-like Disorder After Living Donor Liver Transplantation: A Case Report

BackgroundThrombotic microangiopathy (TMA) pathogenesis after living donor liver transplantation (LDLT) is thought to be caused by release of unusually large von Willebrand factor multimers (UL-vWFMs) resulting from sinusoidal endothelial cell damage and induction of platelet adhesion and aggregation. A decrease in a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13 (ADAMTS-13) that cleave UL-vWFMs might cause excessive UL-vWFMs activity and result in platelet thrombus formation. However, this phenomenon has not undergone a full pathologic assessment. ProceduresA 60-year-old man was diagnosed with hepatitis C-related end-stage cirrhosis. His son was the donor, and he underwent LDLT. On postoperative day 44, his laboratory findings met most TMA diagnostic criteria, and he was diagnosed with TMA-like disorder (TMALD). Localization of CD42b as a platelet marker, vWF, and ADAMTS-13 in allograft tissue of this patient were evaluated using immunohistochemistry. ResultsCD42b expression was observed as platelet aggregates attached to hepatocytes or within the hepatocyte cytoplasm, a morphology called extravasated platelet aggregation (EPA). vWF expression was observed mainly as deposited compact clusters, and ADAMTS-13 expression resembled distinct dots throughout the liver tissue. ConclusionThese findings suggest that EPA indicated sinusoidal endothelial cell damage followed by detachment, and vWF deposition resulted from UL-vWFM oversynthesis. ADAMTS-13 might be consumed in the allograft tissue to cleave UL-vWFMs, but ADAMTS-13 levels might be insufficient to cleave all the deposited UL-vWFMs. We present the case of an LDLT recipient diagnosed with TMALD using blood tests, which showed the presence of TMA pathogenesis in the allograft.

Von Willebrand Factor-Rich Platelet Thrombi in the Liver Cause Sinusoidal Obstruction Syndrome following Oxaliplatin-Based Chemotherapy

Oxaliplatin-based chemotherapy is widely used to treat advanced colorectal cancer (CRC). Sinusoidal obstruction syndrome (SOS) due to oxaliplatin is a serious type of chemotherapy-associated liver injury (CALI) in CRC patients. SOS is thought to be caused by the sinusoidal endothelial cell damage, which results in the release of unusually-large von Willebrand factor multimers (UL-VWFMs) from endothelial cells. To investigate the pathophysiology of CALI after oxaliplatin-based chemotherapy, we analyzed plasma concentration of von Willebrand factor (VWF) and the distribution of VWFMs in CRC patients. Twenty-three patients with advanced CRC who received oxaliplatin-based chemotherapy with (n = 6) and without (n = 17) bevacizumab were analyzed. CALI (n = 6) and splenomegaly (n = 9) were found only in patients who did not treated with bevacizumab. Plasma VWF antigen (VWF:Ag) and serum aspartate aminotransferase (AST) levels increased after chemotherapy only in patients without bevacizumab. VWFM analysis in patients who did not receive bevacizumab showed the presence of UL-VWFMs and absence of high molecular weight VWFMs during chemotherapy, especially in those with CALI. In addition, plasma VWF:Ag and AST levels increased after chemotherapy in patients with splenomegaly (n = 9), but not in patients without splenomegaly (n = 14). Histological findings in the liver tissue of patients who did not receive bevacizumab included sinusoidal dilatation and microthrombi in the sinusoids. Many microthrombi were positive for both anti-IIb/IIIa and anti-VWF antibodies. Plasma UL-VWFM levels might be increased by damage to endothelial cells as a result of oxaliplatin-based chemotherapy. Bevacizumab could prevent CALI and splenomegaly through inhibition of VWF-rich platelet thrombus formation.

Relevance of ADAMTS13 to liver transplantation and surgery.

A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) specifically cleaves unusually-large von Willebrand factor (VWF) multimers under high shear stress, and down-regulates VWF function to form platelet thrombi. Deficiency of plasma ADAMTS13 activity induces a life-threatening systemic disease, termed thrombotic microangiopathy (TMA) including thrombotic thrombocytopenic purpura (TTP). Children with advanced biliary cirrhosis due to congenital biliary atresia sometimes showed pathological features of TMA, with a concomitant decrease of plasma ADAMTS13 activity. Disappearance of their clinical findings of TTP after successful liver transplantation suggested that the liver is a major organ producing plasma ADAMTS13. In situ hybridization analysis showed that ADAMTS13 was produced by hepatic stellate cells. Subsequently, it was found that ADADTS13 was not merely responsible to development of TMA and TTP, but also related to some kinds of liver dysfunction after liver transplantation. Ischemia-reperfusion injury and acute rejection in liver transplant recipients were often associated with marked decrease of ADAMTS13 and concomitant formation of unusually large VWF multimers without findings of TMA/TTP. The similar phenomenon was observed also in patients who underwent hepatectomy for liver tumors. Imbalance between ADAMTS13 and VWF in the hepatic sinusoid might cause liver damage due to microcirculatory disturbance. It can be called as "local TTP like mechanism" which plays a crucial role in liver dysfunction after liver transplantation and surgery.

HSCT-Associated Hepatic VOD Is Initiated With Preceding Appearance Of Unusually Large Von Willebrand Factor Multimers In Patient Plasmas

Backgrounds and AimsHepatic veno-occlusive disease (VOD) associated with hematopoietic stem cell transplantation (HSCT) is characterized by a clinical triad of jaundice (total bilirubin, >2 mg/mL), hepatomegaly with right upper quadrant pain, and ascites and/or unexplained body weight gain (>5% of baseline) within 30 days after operation. Although the pathogenesis of hepatic VOD has not been fully elucidated, the common pathological features are thrombi formed in hepatic central vein.We previously reported that a significant decrease of plasma ADAMTS13 activity was noted in patients undergoing HSCT, who subsequently developed VOD (Park et al, BMT 2002). Then, we showed that plasma antigen levels of von Willebrand factor (VWF) has been kept higher in HSCT-patients with VOD than in those without, and in fact prophylactic infusions of fresh frozen plasma (FFP) with a dose of 10 ml/kg body weight 3 times per week were effective to reduce the frequency of VOD occurrence in high risk patients (Matsumoto et al, BMT 2007). However, more recent studies by ours indicate that FFP infusion alone is not enough to totally eliminate the occurrence (unpublished). Recently, it has been shown that the treatment with recombinant soluble thrombomodulin (rTM) is sometimes highly efficient to reverse VOD progression. But its pharmacokinetics and regimen for the treatment has not been established. As a first step to elucidate a possible combination regimen of FFP and rTM to VOD patients, here we have analyzed the transitional changes of unusually large VWF multimers (UL-VWFMs). The UL-VWFMs are released from damaged endothelial cells and induce platelet hyperagglutination under high shear stress generated in microvasculatures, and are often observed in patient plasmas undergoing HSCT. Patients and MethodsDuring 2011-2012, 45 patients were received allogenic HSCT in the second internal medicine department of our university hospital. None of these patients ,however, were received planned prophylactic FFP infusions, and as a result six patients undergoing allogenic cord blood transplantation (CBT) developed VOD. Clinical features of these 6 patients are shown in Table 1. [Display omitted] Under approval of Ethics Committee of Nara Medical University, we consecutively collected patient's citrated plasmas (ca 2.5 ml) and stored at -80°C until use. Using these deep-frozen plasma samples, we here extensively analyzed plasma levels of ADAMTS13 activity (ADAMTS13:AC), VWF antigen (VWF:Ag), and VWF collagen binding activity (VWF: CBA), together with VWF multimer analysis. More importantly, we also measured the corrected platelet count increment (CCI) to evaluate the efficiency of Platelet trsnsfusions. Then, we comprehensively evaluated these data with routine clinical and laboratory findings. Results and Discussion1) Plasma levels of ADAMTS13:AC were moderately but consistently decreased during two months after HSCT, whereas those of VWF:Ag were kept high, usually more than 200% and often 500% of the normal. 2) The UL-VWFMs appeared soon after HSCT, and continued at least until absolute neutrophil count (ANC) increased to >500 (usually 20-30 days after HSCT). 3) Until platelet engraftment (usually 40-60 days after HSCT), platelet transfusions (every 2-3 days interval) are usually performed to prevent the bleeding complications. During that period, the CCI values were consistently low, but those values were significantly increased during the administration of rTM. 4) Excess platelet transfusions before platelet engraftment induced the consumption of larger VWFMs in patient's plasmas, and often almost simultaneously hepatic VOD developed. Thus, platelet transfusions during the appearance of UL-VWFMs in patient's plasmas may induce platelet clumping in microvasculatures, and lead to the development of thrombotic complications including hepatic VOD. 5) The measurement of plasma levels of VWF:CB activity appeared to well predict the presence of UL-VWFMs. A representative case is shown in Figure. [Display omitted] Thus, a combination regimen of FFP and rTM might be advisable when the patients show the early clinical signs of hepatic VOD and the laboratory data such as VWF:CBA suggest the presence of UL-VWFMs in patient's plasmas. Disclosures:Matsumoto:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Fujimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees; Baxter International Inc: Membership on an entity's Board of Directors or advisory committees.

A Long-Term Phenotype Analysis Of 51 Patients With Upshaw–Schulman Syndrome In Japan, With Special References To Pregnancy and Renal Failure That Requires Hemodialysis

Backgrounds and AimsThe classic hallmarks of Upshaw Schulman syndrome (USS) are severe newborn jaundice with a negative Coombs test that requires an exchange transfusion and repeated childhood episodes of thrombocytopenia and microangiopathic hemolytic anemia that are reversed by infusions of fresh frozen plasma. But now, it is well established that USS is a hereditary deficiency in the activity of von Willebrand factor (VWF)-cleaving protease, termed ADAMTS13. The inheritance mode is autosomal recessive, and their parents are usually asymptomatic carriers having one disease-causing mutation (DCM) of the gene.In the absence of ADAMTS13, unusually large VWF multimers (UL-VWFMs) released from vascular endothelial cells are inappropriately cleaved, leading to platelet hyperagglutination under high shear stress. Thus, USS is alternatively called congenital thrombotic thrombocytopenic purpura (TTP). But, unlike acquired TTP, clinical signs of USS patients are usually mild during childhood and often an isolated thrombocytopenia is found. Further, now it is known that TTP-bouts are aggravated or initiated by the following factors: 1) severe infections such as influenza, 2) pregnancy, 3) 1-deamino-8-D-arginine vasopressin (DDAVP) administration, 4) interferon therapy, 5) heavily drinking alcohol, and 6) aging.So far, in worldwide approximately 150 patients with USS have been found, of which we identified 51 patients in Japan. Among them, 48 patients were received ADAMTS13 gene analysis, and a pair of DCMs, either homozygotes or compound heterozygotes, was identified in all patients except for one. Interestingly, these DCMs are quite different from those found in patients of Western countries and United States, but several DCMs in Japanese patients are also found in Korean and Chinese patients. Thus, it appears that Caucasians and Asians have two different DCM routs for ADAMTS13. Further, since USS is an extremely rare disease, no one can predict the life-long clinical outcome in these patients. Thus, in this study using our cohort of 51 Japanese USS patients, we have extensively analyzed a long-term phenotype, with special references to pregnancy and renal failure that requires hemodialysis. Methods and PatientsADAMTS13 activity and its neutralizing antibodies (inhibitors) were determined by chromogenic ADAMTS13-act-ELISA [Kato et al. Transfusion, 2006], and the IgG type binding antibodies were assayed as described [Ferrari et al, JTH 2009]. Fifty-one patients with USS (19 males and 32 females, born in 1931-2013) were enrolled in this study. None of the patients had the neutralizing antibodies, but 6 patients (6/51, 12%) developed the IgG type non-neutralizing antibodies. Results and DiscussionPregnancy: We identified 26 episodes of pregnancy in 15 patients with USS (Table 1). Briefly, 22 pregnancies were identified before a diagnosis of USS, and as consequence 3 episodes are related to abortion, 10 episodes to stillbirth, and 9 episodes to live birth, in which 6 babies were premature. In contrast, 4 pregnancies were after a diagnosis of USS, and they all had the planned FFP infusions from the early phase of pregnancy, resulting in all successful deliveries, but with 2 premature babies.Renal failure: We identified that 6 male patients finally fell into the end-stage severe renal diseases that required hemodialysis, of which 4 patients were deceased (Table 2). Median time from a diagnosis of USS to dialysis initiation was 15 years (range 1-25). Curiously, all these patients except for one had received FFP infusions biweekly after a diagnosis of USS. Causes of death in these 4 patients were the followings: heart failure (n=1), renal failure (n=1), and sudden death (n=2). The reason why all the patients, who fell into severe renal dysfunction, are male left unaddressed. [Display omitted] [Display omitted] Disclosures:Fujimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees; Baxter BioScience: Membership on an entity's Board of Directors or advisory committees. Matsumoto:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.

Ticlopidine‐associated ADAMTS13 activity deficient thrombotic thrombocytopenic purpura in 22 persons in Japan: a report from the Southern Network on Adverse Reactions (SONAR)

Ticlopidine‐associated <scp>ADAMTS</scp>13 activity deficient thrombotic thrombocytopenic purpura in 22 persons in Japan: a report from the Southern Network on Adverse Reactions (<scp>SONAR</scp>)

Anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a disorder caused by excessive platelet aggregation in multiple organs. Unless the patients are treated with plasma exchange, this disorder leads to early death. Recent studies show that TTP is caused by deficiency of a plasma metalloprotease ADAMTS13, which specifically cleaves von Willebrand factor (VWF). In the absence of ADAMTS13, unusually large VWF multimers (UL-VWFMs) released from endothelial cells are not cleaved appropriately, and cause platelet-rich microvascular thrombosis under high shear stress. Deficiency of ADAMTS13 is caused by autoantibodies against ADAMTS13 in patients with acquired TTP and mutations of the ADAMTS13 gene in congenital TTP. ADAMTS13 antibodies may inhibit enzymatic function or clear ADAMTS13 from circulation. Anti-ADAMTS13 antibodies are comprised predominantly of immunoglobulin class G (IgG). Epitope mapping studies showed that antibodies direct towards the spacer domain of ADAMTS13 are present in most patients with acquired TTP. The Spacer domain contributes to the binding of ADAMTS13 to unfolded VWF. Plasma exchange therapy for acquired TTP is effective because of removing ADAMTS13 autoantibodies and UL-VWFMs, and supply of ADAMTS13. In addition to plasma exchange, corticostroids are usually used for reducing the production of anti-ADAMTS13 antibodies. Recent studies have shown benefit in using rituximab as a first line therapy of acute acquired TTP.

In vivo imaging analysis of the interaction between unusually large von Willebrand factor multimers and platelets on the surface of vascular wall

To elucidate how unusually large von Willebrand factor (UL-VWF) multimers facilitate thrombus formation, their behavior was analyzed together with that of platelets in living mice deficient in the gene encoding the protease that cleaves UL-VWF, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13-/-). By crossing ADAMTS13-/- mice with green fluorescent protein-expressing transgenic mice (GFP mice), GFP-ADAMTS13-/- mice were obtained. The dynamics of GFP-expressing platelets were monitored employing intravital confocal fluorescent microscopy. Administration of a vasopressin derivative, DDAVP, a secretagogue of VWF increased the number of platelets adhered to vascular endothelial cells (VECs) on mesentery at sites recognized by an anti-VWF antibody. Some of these platelets were interconnected and aligned as beads on a string. They reached their maximum length at 5 min and were longer in GFP-ADAMTS13-/- mice than in GFP mice (5.3 ± 4.3, N = 6 vs 2.9 ± 2.1 μm, N = 4) (mean±SE). Focal injury of VECs by topical application of FeCl(3) developed longer (25, 3-50 vs 10, 2-25 μm, P < 0.01) (mean, 10th-90th percentile) and more stable (1.3, 0.3-6.3 vs 0.3, 0.2-1.3 s, P < 0.01) connected platelets in GFP-ADAMTS13-/- mice than in GFP mice. This study revealed that ADAMTS13 cleaves platelet-bound UL-VWF multimers, both during their secretion from VECs and after their adherence to injured vascular walls in veins. UL-VWF multimers either being secreted from VECs or circulating in plasma of ADAMTS13-/- mice appeared to facilitate the accumulation of longer and more stable VWF strings with more associated platelets on injured vascular walls.

Determination of ADAMTS13 and Its Clinical Significance for ADAMTS13 Supplementation Therapy to Improve the Survival of Patients with Decompensated Liver Cirrhosis.

The liver plays a central role in hemostasis by synthesizing clotting factors, coagulation inhibitors, and fibrinolytic proteins. Liver cirrhosis (LC), therefore, impacts on both primary and secondary hemostatic mechanisms. ADAMTS13 is a metalloproteinase, produced exclusively in hepatic stellate cells, and specifically cleaves unusually large von Willebrand factor multimers (UL-VWFM). Deficiency of ADAMTS13 results in accumulation of UL-VWFM, which induces platelet clumping or thrombi under high shear stress, followed by sinusoidal microcirculatory disturbances and subsequent progression of liver injuries, eventually leading to multiorgan failure. The marked imbalance between decreased ADAMTS13 activity (ADAMTS13 : AC) and increased production of UL-VWFM indicating a high-risk state of platelet microthrombi formation was closely related to functional liver capacity, hepatic encephalopathy, hepatorenal syndrome, and intractable ascites in advanced LC. Some end-stage LC patients with extremely low ADAMTS13 : AC and its IgG inhibitor may reflect conditions similar to thrombotic thrombocytopenic purpura (TTP) or may reflect “subclinical TTP.” Hence, cirrhotic patients with severe to moderate deficiency of ADAMTS13 : AC may be candidates for FFP infusion as a source of ADAMTS13 or for recombinant ADAMTS13 supplementation. Such treatments may improve the survival of patients with decompensated LC.

H1N1 Influenza (Swine Flu)-Associated Thrombotic Microangiopathy with a Markedly High Plasma Ratio of von Willebrand Factor to ADAMTS13

We describe an 18-year-old woman infected with H1N1 influenza followed by thrombotic microangiopathy. During the acute phase, her plasma levels of von Willebrand factor (VWF) were remarkably elevated, whereas those of ADAMTS13 were reduced without its inhibitors, generating a markedly high ratio of VWF to ADAMTS13 in circulation. A retrospective analysis established the following hypothesis: an influenza-mediated cytokine storm induced an enhanced release of unusually large VWF multimers (UL-VWFM) from vascular endothelial cells, generating platelet thrombi in microcirculatures under high shear stress. Plasma exchange removed UL-VWFM and cytokines, and rescued her life. This report sheds a light on a hitherto unrecognized influenza complication.

Potential Role of Enhanced Cytokinemia and Plasma Inhibitor on the Decreased Activity of Plasma ADAMTS13 in Patients With Alcoholic Hepatitis: Relationship to Endotoxemia

Deficiency of ADAMTS13 (adisintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13) results in an increase in unusually large von Willebrand factor multimer (UL-VWFM) of the plasma and finally causes microcirculatory disturbance. Our previous study demonstrated that the imbalance of increased UL-VWFM over decreased ADAMTS13 activity may contribute to the development of multiorgan failure in patients with alcoholic hepatitis (AH). The aim of this study was to explore the potential mechanism to reduce the activity of plasma ADAMTS13. Plasma cytokine levels including interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha), plasma endotoxin concentration, and the plasma inhibitor against ADAMTS13 were determined together with ADAMTS13 activity, VWF antigen (VWF:Ag), and UL-VWFM in 24 patients with AH and 5 patients with severe alcoholic hepatitis (SAH). The concentrations of IL-6, IL-8, and TNF-alpha on admission were significantly higher in patients with SAH than in those with AH and controls. The ADAMTS13 activity concomitantly decreased, and the VWF:Ag progressively elevated with increasing concentrations of these cytokines from normal range to over 100 pg/ml. Plasma endotoxin concentration was markedly higher in patients with SAH (mean 52.3 pg/ml) and AH (21.7 pg/ml) than in controls (7.9 pg/ml). The endotoxin concentration inversely correlated with ADAMTS13 activity and was higher in patients with UL-VWFM than those without. The inhibitor was detected in 4 patients with SAH (0.9 to 2.1 BU/ml) and 6 patients with AH (0.5 to 1.6 BU/ml). Patients with the inhibitor showed lower functional liver capacity, higher endotoxin concentration, and marked inflammatory signs than those without. At the recovery stage, the ADAMTS13 activity increased to normal range, the VWF:Ag decreased, and the UL-VWFM disappeared with the decrease in the concentrations of cytokines and endotoxin, and the disappearance of the inhibitor. Decreased ADAMTS13 activity and increased VWF:Ag could be induced not only by pro-inflammatory cytokinemia, but also by its inhibitor, both of which may be closely related to enhanced endotoxemia in patients with AH and SAH.

Pivotal role of ADAMTS13 function in liver diseases

The liver is a major source of clotting and fibrinolytic proteins, and plays a central role in thrombo-regulation. Patients with advanced liver diseases tend to bleed because of reduced plasma levels of several clotting factors and thrombocytopenia, but they do also exhibit thrombotic complications. ADAMTS13 is a metalloproteinase, produced exclusively in hepatic stellate cells, and specifically cleaves highly multimeric von Willebrand factor (VWF). VWF plays a pivotal role in hemostasis and thrombosis, and its function is dependent on its multimeric state. Deficiency of ADAMTS13 results in accumulation of unusually large VWF multimers (UL-VWFM) in plasma, in turn induces platelet clumping or thrombi under high shear stress, followed by microcirculatory disturbances. Considering that UL-VWFM, the substrate of ADAMTS13, is produced in transformed vascular endothelial cells at sites of liver injury, decreased ADAMTS13 activity may be involved in not only sinusoidal microcirculatory disturbances, but also subsequent progression of liver injuries, eventually leading to multiorgan failure. This concept can be applied to the development or aggravation of liver diseases, including liver cirrhosis, alcoholic hepatitis, veno-occlusive disease, and adverse events after liver transplantation. These results promise to bring further understanding of the pathophysiology of liver diseases, and offer new insight for development of therapeutic strategies.