And Metalloproteinase With Thrombospondin Type 1 Motifs 13 Research Articles

Clinical Manifestations, Current and Future Therapy, and Long-Term Outcomes in Congenital Thrombotic Thrombocytopenic Purpura.

Congenital thrombotic thrombocytopenic purpura (cTTP) is an extremely rare disease characterized by the severe deficiency of a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13), caused by ADAMTS13 mutations. While ADAMTS13 supplementation by fresh frozen plasma (FFP) infusion immediately corrects platelet consumption and resolves thrombotic symptoms in acute episodes, FFP treatment can lead to intolerant allergic reactions and frequent hospital visits. Up to 70% of patients depend on regular FFP infusions to normalize their platelet counts and avoid systemic symptoms, including headache, fatigue, and weakness. The remaining patients do not receive regular FFP infusions, mainly because their platelet counts are maintained within the normal range or because they are symptom-free without FFP infusions. However, the target peak and trough levels of ADAMTS13 to prevent long-term comorbidity with prophylactic FFP and the necessity of treating FFP-independent patients in terms of long-term clinical outcomes are yet to be determined. Our recent study suggests that the current volumes of FFP infusions are insufficient to prevent frequent thrombotic events and long-term ischemic organ damage. This review focuses on the current management of cTTP and its associated issues, followed by the importance of upcoming recombinant ADAMTS13 therapy.

The Ratio of von Willebrand Factor Antigen to ADAMTS13 Activity: Usefulness as a Prognostic Biomarker in Acute-on-Chronic Liver Failure

Acute-on-chronic liver failure (ACLF) has a high risk of short-term mortality. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF). Imbalance between ADAMTS13 and VWF is associated with portal hypertension, which induces ACLF development. A previous study reported that ADAMTS13 activity (ADAMTS13:AC) and VWF antigen (VWF:Ag) are predictive biomarkers of ACLF development in patients with cirrhosis. This study investigated the changes in ADAMTS13:AC and VWF:Ag levels from before to after the development of ACLF to determine their usefulness as a prognostic biomarker in patients with ACLF. In total, 101 patients with cirrhosis were enrolled in this study. The level of ADAMTS13:AC and VWF:Ag was determined by an enzyme-linked immunosorbent assay. Cox proportional hazard regression analysis was conducted to determine independent prognostic factors for patients with liver cirrhosis in the post-ACLF group. ADAMTS13:AC levels gradually decreased in the order of non-ACLF group, pre-ACLF group, and finally post-ACLF group. VWF:Ag and the ratio of VWF:Ag to ADAMTS13:AC (VWF:Ag/ADAMTS13:AC) levels gradually increased in the order of non-ACLF group, pre-ACLF group, followed by post-ACLF group. VWF:Ag/ADAMTS13:AC and CLIF-C ACLF scores were associated with prognosis in the post-ACLF group in multivariate analysis. The cumulative survival of the post-ACLF group was significantly lower for patients with high VWF:Ag/ADAMTS13:AC (>9) compared with those with low VWF:Ag/ADAMTS13:AC (≤9) (HR: 10.72, 95% confidence interval: 1.39-82.78, p < 0.05). The VWF:Ag/ADAMTS13:AC increased according to the progression of ACLF in patients with cirrhosis and predicted prognosis in patients with cirrhosis with ACLF.

Comparison of VWF-ADAMTS13 axis involvement in atrial fibrillation and sinus rhythm in patients with ST-elevation myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: None. Background Previous studies have shown that von Willebrand factor (VWF) increases, and a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) decreases in myocardial infarction. Few studies have examined the involvement of the VWF-ADAMTS13 axis in ST-elevation myocardial infarction (STEMI) patients with atrial fibrillation (AF). Purpose Our aim was to investigate VWF-ADAMTS13 axis involvement in atrial fibrillation in STEMI. Methods The subjects of this study were participants in an observational study for investigating the difference in the pathology of thrombus retrieved from infarct-related artery between AF and sinus rhythm (SR). From June 2019 to August 2020, patients who underwent thrombus aspiration for STEMI at our hospital. Only subjects whose thrombus could be confirmed were enrolled. They were divided into two groups, the AF group, and the SR group. Serum biomarkers, including ADAMTS13 activity, and VWF were evaluated. Results 34 patients with STEMI who were able to aspirate thrombus were enrolled in the study. They were divided into two groups: 6 patients with AF (AF group, mean age 72.5 years, 100% male) and 28 patients with SR (SR group, mean age 63.0 years, 100% male). ADAMTS13 activity was significantly lower in AF group than SR group (AF: 0.63 ± 0.14 vs. SR: 0.77 ± 0.13 U / mL, p = 0.037). There was no significant difference in VWF antigen between the two groups (AF: 251 ± 56 vs. SR: 204 ± 69%, p = 0.170). VWF/ADAMTS13 activity in AF group were significantly higher than in SR group. Conclusion Our study suggests an association between the VWF-ADAMTS13 axis and atrial fibrillation in STEMI. Abstract Table. Comparison of serum biomarkers

Ratio of von Willebrand factor antigen to ADAMTS13 activity is a useful biomarker for acute-on-chronic liver failure development and prognosis in patients with liver cirrhosis.

Acute-on-chronic liver failure (ACLF) is associated with a high risk of short-term mortality after progression to multiple organ failure. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF). An imbalance between ADAMTS13 enzyme and VWF substrate is associated with liver cirrhosis progression that induces ACLF. This study examined the relationship between ADAMTS13 and VWF and ACLF development to determine whether ADAMTS13 and VWF are useful predictive biomarkers for ACLF development and prognosis of patients with liver cirrhosis. The study enrolled 67 patients with Child-Pugh class A and B liver cirrhosis. ADAMTS13 activity (ADAMTS13:AC) and VWF antigen (VWF:Ag) were measured using enzyme-linked immunosorbent assays. The ratio of VWF:Ag to ADAMTS13:AC (VWF:Ag/ADAMTS13:AC) was used to divide patients into two groups according to the classification and regression tree based on Gray model survival analysis. Compared with patients with Child-Pugh class A liver cirrhosis, class B patients had a higher VWF:Ag/ADAMTS13:AC and a higher risk of ACLF development. Cumulative incidence of ACLF was significantly higher in patients with high (>7.9) versus low (≤7.9) VWF:Ag/ADAMTS13:AC (hazard ratio [HR], 6.50; 95% CI, 2.31-18.29; p<0.001). Cumulative survival was significantly lower in cirrhotic patients with high versus low VWF:Ag/ADAMTS13:AC (HR 5.11; 95% CI, 1.85-14.14; p=0.002). For patients with liver cirrhosis, VWF:Ag/ADAMTS13:AC is associated with functional liver reserve and predicts the development of ACLF and the prognosis.

Pregnancy-onset thrombotic thrombocytopenic purpura with nephrotic syndrome: a case report

Pregnancy-onset thrombotic thrombocytopenic purpura (TTP) was reported by many obstetricians and hematologists, but less by nephrologists, and the detailed clinical course of its renal complication is not known. Here, we report a case of a 33-year-old pregnant woman who suffered from pregnancy-onset TTP with nephrotic syndrome which was controlled by the termination of pregnancy. On admission, she had periorbital and lower leg edema at 32weeks of gestation. Her serum albumin level was 2.8g/dL and the urine protein/creatinine ratio was 4.1g/g Cr. Besides those, she had thrombocytopenia, hemolytic anemia, and severe deficiency of A Disintegrin-like and Metalloproteinase with Thrombospondin type 1 motifs 13 (ADAMTS-13) activity. Thus, she was diagnosed with nephrotic syndrome due to pregnancy-onset TTP. A cesarean section was performed without complications for the patient and her baby. Then, all her symptoms improved shortly. She was suspected of congenital TTP because of no ADAMTS-13 inhibitor results and the persistent deficiency of ADAMTS-13 activity even after her condition improved. Pregnancy-onset TTP can cause nephrotic syndrome. Termination of pregnancy should be considered in cases with pregnancy-onset TTP to protect kidney function.

Acquired Thrombotic Thrombocytopenic Purpura Without Anti-ADAMTS13 Antibody Caused by Influenza A (H1N1) Virus Successfully Treated by Plasma Exchange: A Case Report

Patient: Female, 57-year-oldFinal Diagnosis: Acquired thrombocytopenic purpura • influenza A virus infectionSymptoms: HypoxemiaMedication:—Clinical Procedure: Plasma exchangeSpecialty: Critical Care Medicine • HematologyObjective:Rare diseaseBackground:Thrombotic microangiopathy, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage by microvascular thrombosis, has a high mortality rate; therefore, early diagnosis and treatment are important. Thrombotic thrombocytopenic purpura is caused by a deficiency of a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13), and results in thrombotic microangiopathy. Influenza virus causes thrombotic thrombocytopenic purpura by inducing immunoglobulin G autoantibodies against ADAMTS13. We report a rare case of thrombotic thrombocytopenic purpura caused by influenza A without anti-ADAMTS13 antibody that was treated by plasma exchange.Case Report:A 57-year-old woman was admitted to our hospital because of hypoxemia. We diagnosed pneumonia and disseminated intravascular coagulation. Despite treatment, she developed thrombocytopenia, and we diagnosed thrombotic microangiopathy and started plasma exchange. With a PLASMIC score of 6 points and neuropsychiatric symptoms, we strongly suspected thrombotic thrombocytopenic purpura and started rituximab. However, ADAMTS13 activity by FRETS-VWF73 assay was 65%, and anti-ADAMTS13 antibody was negative. After 4 plasma exchanges and 2 rounds of rituximab, platelet numbers and lactate dehydrogenase and creati-nine concentrations normalized on the 16th day of hospitalization. Subsequently, influenza A (H1N1) was identified in a nasopharyngeal swab collected on admission. Plasma enzyme-linked immunosorbent assay testing for chromogenic ADAMTS13 activity showed a significant decrease (<0.5%). Therefore, we diagnosed thrombotic thrombocytopenic purpura caused by influenza A without anti-ADAMTS13 antibody.Conclusions:We present a rare case of thrombotic thrombocytopenic purpura without anti-ADAMTS13 antibody caused by influenza A virus successfully treated by plasma exchange. Influenza A may reduce ADAMTS13 activity without inducing autoantibodies.

Predictive Ability of Preoperative PT-INR and Postoperative MCP1 for Post-hepatectomy Liver Failure.

We sought a diagnostic tool using perioperative variables that might predict post-hepatectomy liver failure (PHLF). In 68 patients undergoing major hepatectomy, data on inflammatory markers and coagulation factors were prospectively collected and were compared between patients with International Study Group of Liver Surgery definition grade B/C PHLF (LF group) and those without LF (non-LF group). Preoperatively, the LF group (n=9; 13.2%) had a lower platelet count and a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) activity and a higher prothrombin time-International Normalized Ratio (PT-INR) than the non-LF group. On postoperative day 1, the LF group had significantly higher serum interleukin 6 (IL6), C-C motif chemokine ligand 2 (CCL2), and IL10 levels than the non-LF group. The logistic regression model that included preoperative PT-INR and CCL2 on postoperative day 1 predicted grade B/C PHLF with 100% sensitivity and 89.8% specificity. Our findings suggest that the combination of preoperative PT-INR and CCL2 on postoperative day 1 can predict PHLF earlier and precisely after major hepatectomy.

ADAMTS13 and von Willebrand factor are useful biomarkers for sorafenib treatment efficiency in patients with hepatocellular carcinoma.

BACKGROUNDMany advanced hepatocellular carcinoma (HCC) patients are receiving sorafenib treatment. Sorafenib reportedly improves overall survival (OS) significantly in patients with HCC. Prediction of sorafenib response and prognosis in patients with HCC receiving sorafenib treatment are important due to the potentially serious side effects of sorafenib. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) and von Willebrand factor (VWF) are associated with the pathophysiology of liver cirrhosis and HCC through their roles in hypercoagulability; they are also associated with angiogenesis via vascular endothelial growth factor (VEGF). The imbalance between ADAMTS13 and VWF was associated with prognosis of various cancers in patients undergoing chemotherapy.AIMTo investigate ADAMTS13 and VWF as potential biomarkers for sorafenib response and prognosis in patients with HCC receiving sorafenib treatment.METHODSForty-one patients with HCC receiving sorafenib treatment were included in this study. The initial daily sorafenib dose was 400 mg in all patients. ADAMTS13 activity (ADAMTS13:AC), VWF antigen (VWF:Ag), VEGF levels were determined by enzyme-linked immunosorbent assay. Univariate and multivariate analyses were used to determine predictive factors for sorafenib response and prognosis in patients with HCC receiving sorafenib treatment.RESULTSADAMTS13:AC was significantly higher in patients with stable disease (SD), partial response (PR), and complete response (CR) than in those with progressive disease (PD) (P < 0.05). In contrast, VWF:Ag and the VWF:Ag/ADAMTS13:AC ratio were significantly lower in patients with SD, PR, and CR than in those with PD (P < 0.05 for both). Multivariate analysis showed that the VWF:Ag/ADAMTS13:AC ratio was the only predictive factor for sorafenib response and ADAMTS13:AC was the only prognostic factor in patients with HCC receiving sorafenib treatment. The patients with a low ADAMTS13:AC (< 78.0) had significantly higher VEGF levels than those with a high ADAMTS13:AC (≥ 78.0) (P < 0.05).CONCLUSIONThe VWF:Ag/ADAMTS13:AC ratio and ADAMTS13:AC are potentially useful biomarkers for sorafenib response and prognosis, respectively, in patients with HCC receiving sorafenib treatment.

Major Changes of von Willebrand Factor Multimer Distribution in Cirrhotic Patients with Stable Disease or Acute Decompensation.

Background There is an unstable balance between pro- and anti-haemostatic processes in patients with cirrhosis. We hypothesized, that in patients with acute decompensation (AD) the major alterations of von Willebrand factor (VWF) could contribute to the pro-thrombotic situation as compared to patients with stable (ST) cirrhosis. Patients and Methods We analysed different parameters of VWF, including detailed multimer distribution by densitometry and platelet adhesion, together with a d isintegrin-like a nd m etalloproteinase with t hrombo s pondin type-1 motifs 13 (ADAMTS13) activity and antigen and C-reactive protein (CRP) levels in patients with ST cirrhosis ( n = 99), with AD ( n = 54) and controls ( n = 92). Results VWF antigen, ristocetin co-factor as well as collagen-binding activities were elevated in both cirrhotic groups in a stepwise manner. There was a decrease in high and an increase in low molecular weight multimer ratios in the majority of ST cirrhosis. However, in 24 out of 54 AD patients, ultra-large VWF multimers (ultra-large molecular weight multimers [ULMWM]) were found. ADAMTS13 activity in ST and AD patients without ULMWM was similar to controls (median [interquartile range; IQR]%: 98 [67–132] and 91 [60–110] vs. 106 [88–117], respectively). The presence of ULMWM in AD patients was associated with low ADAMTS13 activity [33 (24–49)%] and high CRP level [23 (7.1–83.6) mg/L]. Adhesion of normal platelets showed a stepwise increase in the presence of cirrhotic plasmas, reaching the highest level in AD patients with ULMWM. Conclusion Characteristic changes of VWF parameters are seen in ST cirrhosis. In AD patients, highly increased VWF and reduced ADAMTS13 activity could be found, along with the presence of ULMWM, which are possible markers and contributors of the disease progression.

ADAMTS13 missense variants associated with defective activity and secretion of ADAMTS13 in a patient with non-cirrhotic portal hypertension.

Non-cirrhotic intrahepatic portal hypertension (NCIPH) is characterized by thrombotic microangiopathy of the portal venous system, low ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13), and high vWF (von Willebrand factor) levels. This study aimed to screen for ADAMTS13 mutations, focusing on the CUB domain, in these patients. Prospectively recruited NCIPH patients and healthy volunteers underwent tests for plasma vWF-ADAMTS13 balance. Sanger sequencing of the CUB domain of ADAMTS13 was done in a subset of the NCIPH patients, and the detected mutation was screened for in all the study participants. Next-generation sequencing of clinically relevant exome and liver immunostaining for ADAMTS13 was done in patients with detected ADAMTS13 mutation. Plasma vWF-ADAMTS13 balance was significantly altered in 24 NCIPH patients (Child's class A:23, B:1) as compared to 22 controls. On initial sequencing of the CUB domain (17 cases and 3 controls), one NCIPH patient showed a rare missense variant (SNV) at position c.3829C>T resulting in p.R1277W (rs14045669). Subsequent RFLP analysis targeted to the R1277W variant did not detect this in any other NCIPH patient, nor in any of the 22 controls. The NCIPH patient with the R1277W variant had severe ADAMTS13 deficiency, consistently high vWF, other missense SNVs in ADAMTS13, vWF, and complement genes. Immunostaining of his liver biopsy revealed globules of ADAMTS13 within stellate cells. We report missense variants in ADAMTS13, vWF, and complement genes in a patient with NCIPH who had decreased secretion and activity of ADAMTS13 protein. Further studies are needed in NCIPH patients in this regard.

Glucose fluctuation and the resultant endothelial injury are correlated with pancreatic β cell dysfunction in patients with coronary artery disease.

We evaluated whether glucose fluctuation (GF) causes vascular endothelial injury and affects glucometabolic factors during lengthy oral glucose tolerance test (OGTT). We enrolled consecutive 116 patients with coronary artery disease (CAD) who were performed coronary angiography and 4-h OGTT. Blood samples were collected before and 4h after glucose load to measure endothelial injury factor [von Willebrand factor (vWF) and vWF/a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS-13) ratio]. GF was defined as maximum - minimum blood glucose levels during 4-h OGTT. We estimated the relationship between GF and glucometabolic factors. vWV and vWF/ADAMTS-13 ratio were significantly correlated with GF during 4-h OGTT. GF was significantly correlated with homeostasis model to assess insulin resistance (HOMA-IR) (R=0.262), Matsuda index (R=-0.405), insulinogenic index (R=-0.336), HbA1c (R=0.281) and disposition index (R=-0.672). When dividing patients into impaired and preserved category groups according to the average value of GF (122mg/dL), adjusted to age, sex, HOMA-β, insulinogenic index, HOMA-IR, Matsuda index and HbA1c, disposition index was an independent risk factor for impaired GF [odds ratio (95% confidence interval): 2.87 (1.70-4.83), P<0.001]. Pancreatic β cell dysfunction is associated with GF and causes endothelial injury in CAD patients.

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.

Acute myocardial infarction as the initial thrombotic event of thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by the coemergence of microangiopathic hemolytic anemia, thrombocytopenia, and thrombosis-mediated ischemic injuries of various organs, such as the central nervous system and kidneys. Acute myocardial infarction (AMI) has also occasionally been reported as a complication with TTP as the secondary thrombotic event; however, its emergence as the initial thrombotic event in TTP is extremely rare. This report describes an 80-year-old male patient with acquired TTP, who was affected by AMI without any clinically apparent damage to other organs or abnormal laboratory findings that would be suggestive of TTP at the first presentation. Although AMI was successfully treated by percutaneous coronary intervention (PCI), the patient developed marked thrombocytopenia with acute kidney injury and hemolytic anemia 5 days after PCI. The patient was diagnosed as having acquired TTP based on decreased ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13) below the level of detection and the presence of the inhibitor against ADAMTS13, and eventually died of multiorgan failure due to TTP despite undergoing repeated plasma exchanges and immunosuppressive therapies, including corticosteroid and rituximab. Although caution is often paid to therapy-related thrombocytopenia or renal damage after PCI, that is, those caused by antiplatelet drugs, heparin, or contrast agents, our report alerts us to the presence of TTP as an extremely rare, but underlying cause for AMI that could be subclinical at the initial presentation.

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.

関節リウマチに合併した血栓性血小板減少性紫斑病の一症例におけるa disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13（ADAMTS13）活性および抗体価の経時的推移

関節リウマチに合併した血栓性血小板減少性紫斑病の一症例におけるa disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13（ADAMTS13）活性および抗体価の経時的推移

Distinct Role of von Willebrand Factor Triplet Bands in Glycoprotein Ib-Dependent Platelet Adhesion and Thrombus Formation under Flow

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer have one shorter or longer N-terminal peptide sequence, respectively. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Therefore, platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, whereas preparation B predominantly contained larger triplet bands. Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb but an unchanged affinity for collagen type III when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Thus, VWF forms lacking larger-size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.

ADAMTS13 TSP1 Repeat Domains Promote HUVEC Angiogenesis Via Phosphorylation of VEGFR2.

Abstract 2182The primary role of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, 13) is to cleave unusually large von Willebrand factor (ULVWF) multimers under shear stress. Recently, we reported that ADAMTS13 may be a potent mitogen and chemoattractant, and it modulates angiogenesis in vitro (Microvas Res. 2012, 84, 109–115). However, the structural components and mechanism of ADAMTS13 modulating angiogenesis are not understood. Herein, we report the effect of ADAMTS13 variants on cell proliferation, migration, and, tube formation of human umbilical vein endothelial cells (HUVEC). In addition, we determined the signaling pathways by which ADAMTS13 promotes angiogenesis.ADAMTS13 fragments containing TSP1 repeat (i.e. MDT, MDTCS, TSP1 2–8, TSP1 5–8 plus CUB, and TSP1 2–8 plus CUB) were used in this study. In the proliferation model, TSP1 2–8 at the concentration of 34.6 ng/mL (651 pM) increased endothelial cell proliferation by 267 %. In the chemotaxis assay, TSP1 2–8 at the concentration of 27.7 ng/mL (521 pM) increased endothelial cell migration across a gelatinized polycarbonate membrane by 71 %. Similarly, TSP1 2–8 at the concentration of 55.4 ng/mL (1.0 nM) induced endothelial cell tube formation in Matrigel by 45 %. In all three models, the TSP1 2–8 induced angiogenic responses with similar efficacy to full-length ADAMTS13. MDT and MDTCS fragments did not affect proliferation, migration, or tube formation significantly, as compared to the negative control.To determine the mechanism by which ADAMTS13 induces angiogenesis, we incubated endothelial cells with ADAMTS13 at the concentration of 147 ng/mL (1.0 nM). We showed that ADAMTS13 increased the phosphorylation of VEGFR2, Akt, and, P44/42 MAPK, which may trigger downstream activation to promote cell proliferation and migration. Addition of anti-VEGF antibody in the culture system significantly blocked the ADAMTS13-induced effect, indicating that ADAMTS13 plays a role in promoting angiogenesis by inducing VEGF secretion from endothelial cells (Fig 1).The biological role of ADAMTS13 in angiogenesis was further demonstrated in a chick embryo model. Collagen onplants supplemented with EBM-2 (as negative control), 40 ng/mL VEGF165 (2.1 nM) (as positive control), and 306 ng/mL ADAMTS13 (2.2 nM) were placed on the chorioallantoic membrane of day 8 fertilized white leghorn chicken embryos. Localized and sustained release of VEGF and ADAMTS13 over a course of 72 hours resulted in 8-fold increase in capillary migration into the collagen onplants. Together, our findings suggest that the TSP1 repeats of ADAMTS13 metalloprotease promote angiogenesis by inducing VEGF secretion and VEGFR2 phosphorylation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Role of Von Willebrand Factor Triplet Bands in Glycoprotein Ib-Dependent Platelet Adhesion Under Flow Conditions

AbstractAbstract 3356 Objective:Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer respectively have one shorter or longer N-terminal peptide sequence. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Distribution of VWF triplet bands is significantly altered in some types of VWD, however, the impact of triplet structure on VWF function has not been investigated so far. Methods:Platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition obtained by size exclusion in addition to heparin affinity chromatography were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, while preparation B predominantly contained larger triplet bands. Collagen- and GPIb-binding was determined by surface plasmon resonance (SPR). Platelet adhesion under flow was determined using flow-chamber models. Results:Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb, but an unchanged affinity for collagen type III, when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Conclusion:Thus, VWF forms lacking larger size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow. Disclosures:Schwartz:Octapharma: Employment. Kannicht:Octapharma: Employment. Fuchs:octapharma: Employment. Kröning:octapharma: Employment. Solecka:Octapharma: Employment.

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia–reperfusion injury

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia-reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia-reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia-reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia-reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.

Thrombotic microangiopathy due to multiple autoantibodies related to antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a rare disorder in children. More than half of childhood APS occurs as secondary APS complicated by systemic lupus erythematosus (SLE) and other autoimmune diseases. We encountered a boy with SLE who presented with thrombotic microangiopathy (TMA) due to APS. He was initially diagnosed with SLE and treated with methylprednisolone pulse therapy. However, his renal function rapidly deteriorated. Since poikilocytes were detected, we suspected that his condition was complicated by TMA or APS. Urgent plasma exchange, continuous hemodialysis, and intravenous cyclophosphamide saved the patient and his renal failure ameliorated. A renal biopsy performed at the onset of disease showed multiple microvascular thrombi, diffuse mesangiolysis, and cortical necrosis compatible with TMA. He was positive for anticardiolipin antibody, anti-β2-glycoprotein I antibody, and lupus anticoagulant as well as anti-phosphatidylserine-prothrombin complex IgG antibody (aPS/PT). Anti-a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) antibody was negative and ADAMTS13 activity was normal. The aPS/PT is thrombogenic and is a newly discovered lupus anticoagulant. Childhood TMA due to APS has rarely been reported. To the best of our knowledge this is the first report of pediatric TMA due to APS with positive aPS/PT. Physicians need to be aware of aPS/PT in pediatric APS and/or SLE.