ADAMTS13 Levels Research Articles

Serum ADAMTS-13 Levels as an Indicator of Portal Vein Thrombosis.

Background Coagulation disorders in patients with liver cirrhosis are a common clinical problem. Cirrhosis should be considered a state of impaired blood clotting or an imbalance of the whole coagulation system. Cirrhosis-induced coagulopathy encompasses disturbances in both the procoagulant and anticoagulant systems. This mechanism may promote the development of thrombosis with portal vein thrombosis (PVT), which is considered an obstacle to orthotopic liver transplantation (OLT). We assessed serum ADAMTS-13 levels in patients with decompensated liver cirrhosis, with and without PVT. Material and Methods Serum ADAMTS-13 levels, age, platelet count (PLT), and INR (international normalized ratio) were evaluated in (n = 64) patients with liver cirrhosis either with PVT (group 1, n = 31) or without PVT (group 2, n = 33). The results were compared with those from healthy volunteers (group 3, n = 37). Liver cirrhosis was based on Desmet's classification of chronic hepatitis in liver biopsy stage ≥ 3 or liver elastography F-score ≥ 3. Serum ADAMTS-13 levels were measured with Quantikine® ELISA Human ADAMTS13 Immunoassay, R&D Systems Inc. We used Welch's F-test, Games-Howell, one-way ANOVA, Bonferroni test, and logistic regression to determine whether ADAMTS-13 levels were a predictor that was independent of MELD and Child-Pugh scores. All results (P < 0.05) were considered statistically significant. Results The mean serum ADAMTS-13 level in patients with PVT was significantly lower than that in patients without PVT (P = 0.001) and controls (P = 0.001). The mean serum ADAMTS-13 level in patients without PVT was significantly lower than that in controls (P = 0.001). ADAMTS-13 levels were significantly associated with PVT accounting for the Child-Pugh or MELD score in the logistic regression model. Conclusions Low serum ADAMTS-13 levels can be a useful indicator of portal thrombosis in patients with decompensated liver cirrhosis irrespective of Child-Pugh or MELD scores. Further research is needed to determine whether ADAMTS-13 levels will find use in everyday clinical practice.

D-dimer, Von Willibrand factor, and ADAMTS13 in renal transplant recipients

Introduction Chronic kidney disease and renal transplants are associated with activation of coagulation. Microvascular thrombosis and fibrinolytic disorders have been recognized as main causes of allograft rejection in renal transplant recipients. Aims The aim was to evaluate D-dimer, Von Willibrand factor (VWF), and ADAMTS13 activity plasma levels in renal transplant recipients and investigate the association of these parameters and creatinine plasma levels, estimated glomerular filtration rate (eGFR), and time after transplantation. Participants and methods A total of 40 renal transplant recipients clinically stable at National Institute of Urology and Nephrology, 5–192 months after transplantation were enrolled in the study. Dimer, VWF, and ADAMTS13 activity levels were measured. Result We observed significant higher levels of ADAMTS13 (P = 0.03) in subgroup Cr3 (55.9%) with creatinine greater than 2.0 mg/dl as compared with Cr1 (43.3%) with creatinine less than 1.4 mg/dl and insignificant higher levels of D-dimer and VWF in subgroup Cr3 (566.22 ng/ml and 253.5 IU/dl, respectively) as compared with Cr1 (363.3 ng/ml and 240.6 IU/dl, respectively). We observed also insignificant higher levels of D-dimer, VWF, and ADAMTS13 in subgroup with eGFR less than 60 ml/min/1.73 m2 (478.6 ng/ml, 220.6 IU/dl, and 43.3%, respectively) as compared with eGFR greater than or equal to 60 ml/min/1.73 m2 (322.73 ng/ml, 207.32 IU/dl, and 42%, respectively). There was a weak association between eGFR and D-dimer [odds ratio (OR)=−0.033, P = 0.01] and VWF (OR=−0.053, P = 0.044) and a weak association between creatinine plasma levels (>2.0 mg/dl) with D-Dimer (OR = 0.001, P = 0.003) and VWF (OR = 0.001, P = 0.038). Conclusion D-dimer, VWF, and ADAMTS were weakly associated with creatinine plasma levels and graft function. Other studies with a larger number of renal transplant recipients and from more than one center must clarify the role of hemostatic markers, especially, D-dimer, VWF, and ADAMTS13.

ADAMTS13 deficiency and immunological abnormalities in patients with systemic sclerosis.

IntroductionSystemic sclerosis (SSc) is a chronic autoimmune connective tissue disorder characterized by immunological deviations and generalized microvascular damage.AimTo determine the serum level of the von Willebrand factor cleaving protease (ADAMTS13) in 39 SSc patients and healthy controls.Material and methodsADAMTS13 serum level was determined in 39 SSc patients and 11 healthy controls. Complete history of the patients was recorded and thorough clinical, rheumatological, and dermatological examinations were performed.ResultsThe serum levels of ADAMTS13 were significantly lower in SSc than in normal controls (455.47 ±128 vs. 702.01 ±142 ng/ml, p < 0.00001). However significant correlations among serum ADAMTS 13 levels and organ changes were not found in SSc patients.ConclusionsWe demonstrate a decreased serum level of ADAMTS13 in SSc patients, which may contribute to the vessel microangiopathy observed in systemic sclerosis.

Complement Regulatory Genetic Mutations in the Setting of Autoimmune Thrombotic Thrombocytopenic Purpura: A Case Series.

ObjectiveTo explore the benefits of adding eculizumab for the treatment of refractory autoimmune thrombotic thrombocytopenic purpura (iTTP) with complement dysregulation.Patients and MethodsFrom January 1, 2014, through July 1, 2017, we identified patients with iTTP defined by ADAMTS13 (disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) levels less than 5% and the presence of ADAMTS13 inhibitor. Patients who progressed after receiving standard of care management for iTTP were subjected to a comprehensive evaluation to look for evidence of complement activation. Herein, we share our single-institute experience regarding the clinical course and treatment algorithm for 3 patients with refractory iTTP.ResultsAll the patients had clinical deterioration despite treatment with plasma exchange, corticosteroids, rituximab, and vincristine, which prompted us to look for evidence of complement activation and associated genetic mutations. Complement-related genetic aberrations were present in all 3 patients, who had had different degrees of complement activation. The first 2 patients did not benefit from eculizumab when treatment was started before complete clearance of inhibitors to ADAMTS13. However, they had durable remissions when eculizumab was introduced after clearance of ADAMTS13 inhibitors. The third patient started eculizumab therapy after inhibitor levels were undetectable.ConclusionWe found eculizumab therapy to be effective in all 3 patients. However, its efficacy was prominent only after clearance of antibodies against ADAMTS13 via therapeutic plasma exchange.

Elevated preoperative von Willebrand factor is associated with perioperative thrombosis in infants and neonates with congenital heart disease

Essentials Perioperative thrombosis is a major cause of morbidity and mortality in congenital heart disease. Neonates and infants undergoing repair of congenital heart lesions were prospectively followed. Elevated von Willebrand factor (VWF) to ADAMTS-13 activity ratios typified the postoperative period. Thrombosis was associated with preoperative VWF activity and cryoprecipitate transfusion SUMMARY: Background The surgical repair of congenital heart malformations is frequently complicated by perioperative thrombosis of unclear etiology. An imbalance between von Willebrand factor (VWF) and ADAMTS-13 is an emerging variable in thrombosis. Objectives To describe perioperative changes to VWF, ADAMTS-13 and NETosis, and evaluate clinical and biochemical associations with postoperative thrombosis. Methods Neonates and infants undergoing palliation or definitive surgical repair of congenital heart malformations were recruited (n=133). Preoperative and postoperative plasma levels of VWF, ADAMTS-13 and markers of NETosis were determined. Patients were followed for up to 30days for the occurrence of thrombosis. Univariate and multivariate logistic regression analyses were conducted to identify variables associated with thrombosis. Results We identified significant postoperative increases in VWF activity, VWF level, DNA-histone complexes and cell-free DNA with an overall decrease in ADAMTS-13 activity. Patients experiencing postoperative thrombotic events (9%) were characterized by surgery performed at a lower intraoperative temperature, higher preoperative lactic acid levels, and higher preoperative VWF activity and level. A multivariate logistic regression model identified preoperative VWF activity (odds ratio (OR)8.39 per IUmL-1 , 95% confidence interval [CI]1.73-40.55) and transfusion of cryoprecipitate (OR1.10 per mLkg-1 , 95%CI1.03-1.17) as being associated with thrombosis. Conclusions Pediatric patients undergoing surgical repair of congenital heart malformations are exposed to high levels of VWF with diminished or minimal change to ADAMTS-13 in the immediate postoperative period. Elevated preoperative VWF activity is associated with postoperative thrombosis in pediatric congenital heart disease.

Predictive value of ADAMTS-13 on concealed chronic renal failure in COPD patients.

BackgroundImpaired renal function is often neglected in COPD patients. Considering that COPD patients usually have an ongoing prothrombotic state and systemic inflammation status, we investigated the association among them and explored the predictive value of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), on concealed chronic renal failure (CRF) in COPD patients.MethodsCOPD patients were recruited from the First Affiliated Hospital of Sun Yat-Sen University between January 2015 and December 2016. Control was selected from contemporaneous hospitalized patients without COPD and matched by age and gender at a ratio of 1:1. Estimated glomerular filtration rate (eGFR) was calculated by using the Chronic Kidney Disease Epidemiology Collaboration formula, and all subjects were categorized as having normal renal function (eGFR ≥60 mL min−1 1.73 m−2) and having concealed CRF (normal serum creatinine while eGFR <60 mL min−1 1.73 m−2). Independent correlates of concealed CRF were investigated by logistic regression analysis, and receiver operating characteristic (ROC) curves were used to determine the predictive value of ADAMTS-13.ResultsIn total, 106 COPD and 106 non-COPD patients were finally recruited, and the incidences of concealed CRF were 19.81% and 7.55%, respectively. ADAMTS-13 (odds ratio [OR] =0.858, 95% CI =0.795–0.926), D-dimer (OR =1.095, 95% CI =1.027–1.169), and C-reactive protein (OR =1.252, 95% CI =1.058–1.480) were significantly associated with concealed CRF. Sensitivity and specificity at an ADAMTS-13 cutoff of 318.72 ng/mL were 100% and 81.2%, respectively. The area under the ROC curve was 0.959.ConclusionProthrombotic state and systemic inflammation status might contribute to explaining the high incidence of concealed CRF in COPD, and plasma ADAMTS-13 levels may serve as a strong predictor.

ADAMTS13-von Willebrand factor axis is involved in the pathophysiology of kidney ischaemia-reperfusion injury.

The ADAMTS13-von Willebrand factor (vWF) axis has been suggested to play a critical role in the pathophysiology of ischaemia-reperfusion injury (IRI) in the heart or brain. Therefore, we aimed to investigate whether this axis was involved in the pathophysiology of IRI-induced acute kidney injury. We performed renal IRI in ADAMTS13 knockout (KO) or wild type (WT) mice. Functional and histological kidney damage, and inflammation were compared and the effect of anti-vWF antibodies in ADAMTS13 KO mice was assessed. Following IRI, the blood and kidney ADAMTS13 levels were significantly decreased. vWF expression was significantly upregulated in both the medulla and cortex of injured kidneys as shown by immunohistochemistry and western blot analyses. There was also an increased level of vWF dimers after IRI. In ADAMTS13 KO mice, kidney vWF levels were further increased and this was associated with greater endothelial and epithelial injury compared to WT mice, suggesting an important role of vWF in renal IRI. In addition, the number of Gr-1+ neutrophils was significantly higher in the kidneys of ADAMTS13 KO mice compared to WT mice, whereas F4/80 macrophage numbers were unchanged. In ADAMTS13 KO mice, administration of anti-vWF antibodies after IRI partially reversed renal injury. Our data show that the ADAMTS13-vWF axis is partially involved in the pathophysiology of kidney IRI, suggesting that regulating ADAMTS13- and vWF-dependent mechanisms could have therapeutic potential to limit renal IRI.

Balanced haemostasis with both hypo- and hyper-coagulable features in critically ill patients with acute-on-chronic-liver failure

BackgroundCirrhotic patients have complex haemostatic abnormalities. Current evidence suggests stable cirrhotic (SC) patients have a “re-balanced” haemostatic state. However, limited data exists in acute decompensated (AD) or acute on chronic liver failure (ACLF) patients. MethodsWe utilised thrombin generation analysis, fibrinolysis assessment, and evaluation of haemostatic parameters to assess haemostasis in liver disease of progressive severity. ResultsThe study cohorts were comprised of: SC, n=8; AD n=44; ACLF, n=17; and Healthy Control (HC), n=35. There was a progressive increase across the cohorts in INR (p=0.0001), Factor VIII (p=0.0001) and VWF levels (p=0.0001) and a correspondingly decrease in anti-thrombin (p=0.0001), ADAMTS-13 (p=0.01) and fibrinogen levels (p=0.0001). In the presence of thrombomodulin, thrombin generation was equivalent or significantly higher in all the cohorts compared to HC (p=0.0001). Compared to AD, ACLF had a lower ETP (p=0.002) and thrombin peak (p=0.0001). There was no significant difference across the cohorts in clot lysis time (p=0.07), although compared to HC, AD had a significantly shorter lysis time (p=0.001). ConclusionsOur cohorts, despite significant differences in haemostatic parameters, displayed intact thrombin generation but progressive hypo-functional clot stability and potentially but not universal hyper-functional haemostasis.

Evaluation of a cost-effective ADAMTS13 antigen assay

Thrombotic thrombocytopenic purpura (TTP) is life-threatening and is characterised by platelet deposition in the microvasculature with thrombus formation in particular organs. This results in thrombocytopenia, microangiopathic haemolytic anaemia, kidney failure and neurological symptoms. It is a rare disorder, but can occur in patients infected with the human immunodeficiency virus (HIV). ADAMTS13 is the 13 th member of a disintegrin and metalloprotease with thrombospondin type motifs. Its deficiency causes TTP. Therefore, the measurement of the ADAMTS13 levels in plasma is vital in the diagnosis of TTP and also important in distinguishing it from other thrombotic microangiopathies (TMA’s). Unfortunately, commercial ADAMTS13 antigen assays are expensive for healthcare service providers in developing countries. However, several antibodies and antibody pairs have been produced against ADAMTS13 and are commercially available. In this study, we evaluated an in-house ADAMTS13 antigen assay using two different commercial antibodies and compared the outcomes to that of a commercial ADAMTS13 antigen kit by using the plasma of 40 patients with possible HIV-associated TTP and 40 healthy subjects. The Intra- and inter-assay coefficients of variation were calculated as 8% and 7% respectively. The assay gave linear results between 0.78 to 12.5% ADAMTS13. The limit of detection was 0.2%, and the limit of quantification was 0.8%. The correlation of our assay compared to the commercial test kit was excellent, with a R 2 value of 0.9. In addition, the cost of our ADAMTS13 antigen assay was lower than that of the commercial ADAMTS13 antigen test kit. Our cost-effective in-house ADAMTS13 antigen test also produced reliable results. We therefore recommend that this assay be used to diagnose HIV-associated TTP.

Platelet hyperaggregability is associated with decreased ADAMTS13 activity and enhanced endotoxemia in patients with acute cholangitis.

Insufficient ADAMTS13 activity (ADAMTS13:AC) leads to increased levels of unusually large von Willebrand factor (VWF) multimers and causes microcirculatory disturbance and multiple organ failure (MOF). Endotoxin (Et) triggers the activation of coagulation and cytokine cascades, leading to MOF in severe inflammatory response syndrome. Here, we investigated the potential role of endotoxemia-related ADAMTS13 in acute cholangitis. Twenty-four patients with acute cholangitis, including 7 with severe acute cholangitis, were recruited in this study. The levels of ADAMTS13:AC, VWF antigen (VWF:Ag), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in each patient were determined by enzyme-linked immunosorbent assay, whereas Et levels were determined by Et activity assay (EAA) analysis. The ADAMTS13:AC and VWF:Ag levels were significantly lower and higher, respectively, in patients with acute cholangitis than in controls. The EAA levels were higher in patients with acute cholangitis than in controls, and were inversely correlated with that of ADAMTS13:AC. Patients with severe acute cholangitis had significantly lower ADAMTS13:AC and higher VWF:Ag levels than those with mild to moderate cholangitis. Notably, ADMTS13:AC was directly correlated with platelet counts and inversely correlated with IL-6 levels, and the VWF:Ag/ADAMTS13:AC ratio was directly correlated with IL-8 and TNF-α levels. Imbalance of ADAMTS13:AC and VWF:Ag levels might be associated with severe acute cholangitis, reflecting platelet hyperaggregability. Severe acute cholangitis has severe pathophysiological features and is complicated by endotoxemia and MOF. Notably, this is the first report indicating an association between the levels of ADAMTS13:AC and VWF:Ag and those of EAA and cytokines in acute cholangitis.

Association of ABO blood groups with von Willebrand factor, factor VIII and ADAMTS-13 in patients with lung cancer.

Coagulative and fibrinolytic disorders appear to be associated with the development of lung cancer. The aim of the present study was to determine plasma levels of von Willebrand factor (VWF) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 13 (ADAMTS-13), and factor VIII (FVIII) activity, in association with O and non-O blood groups in patients with lung cancer. Plasma levels of VWF and ADAMTS-13, and FVIII activity were measured in 115 patients with lung cancer and 98 healthy subjects. Phenotyping of the ABO blood groups was also performed for the two groups. Significantly increased VWF levels and FVIII activity, as well as significantly decreased ADAMTS-13 levels, were observed in patients with distant metastasis as compared with those without distant metastasis and the healthy controls. Plasma VWF levels and FVIII activity were significantly increased in subjects with non-O type blood compared with those with type O blood in the two groups. However, a significant decrease in ADAMTS-13 levels was observed only in the control group among those with non-O type blood, compared with those with type O blood. The results of the present study indicate that increased VWF and decreased ADAMTS-13 levels facilitate the invasiveness and metastasis of lung cancer. Non-O blood groups constitute a risk factor for increased VWF and FVIII in plasma. Continued monitoring of VWF and ADAMTS-13 levels, and of FVIII activity in patients with lung cancer with distinct blood groups may help to minimize the incidence of thrombotic events and improve assessment of disease progression.

Abstract 1816: Endogenous ADAMTS-13 regulates angiogenesis in cultured human endothelial cells

Abstract ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis; for example; addition of exogenous ADAMTS-13 enhances endothelial cell (Lee et al. Microvascular Res 2012; 84:109) and glioblastoma cell (Lee et al. Blood 2013; 122:#2306) migration and proliferation. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA. 30pmols of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 90% after 24hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. The knockdown effect was decreased over time: 72hr after the initial knockdown, ADAMTS-13 expression was reduced by only 50%. The effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions were studied. Transfection of HUVEC with 10pmols of ADAMTS-13 siRNA in a 24-well plate resulted in a 21% and 22% decrease in proliferation after 24hr and 48hr incubation, respectively. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24hr after transfection with control siRNA, there was robust cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased expression of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. Activity of the AKT pathway, one of the angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA while ERK, a component of the MAP kinase pathway was not affected upon knockdown of ADAMTS-13. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor. Citation Format: Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. Endogenous ADAMTS-13 regulates angiogenesis in cultured human endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2017-1816

Rituximab prophylaxis to prevent thrombotic thrombocytopenic purpura relapse: outcome and evaluation of dosing regimens.

Acute antibody-mediated thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy with high morbidity and mortality. Rituximab is highly effective as prophylaxis in patients at risk of acute TTP relapse, but the ideal dosing regimen is unknown. A multicenter retrospective cohort study evaluated outcomes of patients given rituximab prophylaxis to prevent TTP relapse. Rituximab was given in 76 episodes to 45 patients (34 women and 11 men). Four once-per-week infusions of standard- (375 mg/m2 [24 episodes]), reduced- (200 mg [19 episodes]), and intermediate- (500 mg [17 episodes]) dose rituximab were given; in the remaining 16 episodes, patients received 100 to 1000 mg rituximab in 1 to 5 doses. Patients were deemed at high risk of TTP relapse on the basis of ADAMTS13 activity dropping to ≤15% from the normal range. Preprophylaxis median ADAMTS13 level was 5% (range, <5% to 17%). Normalization of ADAMTS13 occurred in 78.9% of patients, with 92.1% having at least a partial response (ADAMTS13 ≥30%); 3 patients had no response. Over a median of 15 months (range, 1-141 months), there were only 3 TTP relapses (2 of these subacute) in the reduced dose group. Re-treatment with rituximab occurred in 50% of patient episodes at a median of 17.5 months (range, 9-112 months) after initial prophylaxis. There was a statistically higher rate of re-treatment in the reduced- vs standard-dose group: 0.38 vs 0.17 episodes per year, respectively. Treatment was generally well tolerated, infusional effects being the most commonly reported. Rituximab therapy is effective as prophylaxis for normalizing ADAMTS13 and is an additional measure for preventing acute TTP relapses in patients with immune TTP.

Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the ADAMTS13 locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of ADAMTS13 (β, 6.7%; P = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (β, -22.3%; P = 1.2E-30) and rs3124762 (β, 3.5%; P = 8.9E-9) close to ADAMTS13 and rs4075970 (β, 2.4%; P = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around ADAMTS13 (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous ADAMTS13 variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.

Von Willebrand Factor: Multimeric Structure and Functional Activity in Patients With Atrial Fibrillation With and Without Oral Anticoagulation.

von Willebrand factor (vWF) is a multimeric glycoprotein present in blood plasma. It is synthesized in megakaryocytes and endothelial cells, secreted into circulation in the form of high-molecular-weight multimers (HMWMs), and cleaved into shorter, less active multimers by ADAMTS13. It is essential for platelet adhesion and aggregation. Previous studies have investigated the relationship between vWF levels and thromboembolic events with little regard to vWF multimeric structure. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 levels. One hundred seven patients with AF, 51 anticoagulated and 56 nonanticoagulated, were eligible for the study. Plasma samples were analyzed for vWF antigen, vWF activity, and ADAMTS13; vWF multimers were analyzed by Western blot in 1% to 1.3% sodium dodecyl sulfate agarose gel electrophoresis. Patients with AF without oral anticoagulation (OAC) had significantly higher vWF plasma levels (154.00 [75-201] UI/dL) and vWF activity (60.00% [20%-210%]) compared to patients with OAC (133.50 [90-192] UI/dL, P = <.001; 50.00% [20%-160%], P = .02). Both were specially decreased in patients treated with acenocumarin. Patients without OAC also showed lower ADAMTS13 levels and presence of vWF HMWMs. Patients with AF show higher plasma levels and vWF activity. Moreover, treatment with traditional OAC (acenocumarin) significantly reduced vWF levels. Patients without OAC might have an increased risk of thrombotic events showing lower ADAMTS13 and higher vWF levels. Patients with stroke had higher plasma levels, vWF activity, and HMWMs. Our study suggests that increased vWF levels and presence of HMWMs could be related to cerebrovascular disease and may represent useful biomarkers for stroke in AF.

Reduced ADAMTS13 levels in patients with acute and chronic cerebrovascular disease.

Von Willebrand Factor (VWF) plays a major role in thrombosis and hemostasis and its thrombogenicity is controlled by ADAMTS13. Whereas increasing evidence shows a clear association between VWF levels and acute ischemic stroke, little is known about a correlation with ADAMTS13. Therefore, the aim of this study was to compare plasma levels of ADAMTS13 between 85 healthy volunteers (HV), 104 patients with acute ischemic stroke and 112 patients with a chronic cerebrovascular disease (CCD). In this case-control study, plasma ADAMTS13 antigen levels were measured by ELISA and plasma VWF levels, measured previously, were next used to calculate VWF:ADAMTS13 ratios. ADAMTS13 levels and VWF:ADAMTS13 ratios were subsequently correlated with key demographic and clinical parameters. ADAMTS13 levels were significantly lower in acute ischemic stroke patients (82.6 ± 21.0%) compared with HV (110.6 ± 26.9%). Also, CCD patients (99.6 ± 24.5%) had significantly lower ADAMTS13 levels compared with HV however these were still higher than in acute stroke patients. Furthermore, when assessing the VWF:ADAMTS13 ratios, an even greater difference was revealed between stroke patients (2.7 ± 1.9), HV (1.1 ± 0.5) and CCD patients (1.7 ± 0.7). The VWF:ADAMTS13 ratio was significantly associated with stroke severity and modality. In conclusion, both in acute and chronic cerebrovascular disease patients, ADAMTS13 levels were significantly decreased, with the lowest ADAMTS13 levels found in acute stroke patients. This difference was even more distinct when the ratio of VWF:ADAMTS13 was considered. These results demonstrate the potentially important involvement of the VWF/ADAMTS13 axis in ischemic stroke.

Protective effect of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 haplotype on coronary artery disease.

: Genetic variations of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and von Willebrand factor (vWF) were related to ADAMTS13 levels. Reduction of ADAMTS13 activity may affect atherosclerotic progression. However, the associations of polymorphisms of these genes with coronary artery disease (CAD) are still unclear. This study, therefore, aimed to investigate the relationship of genetic variations and haplotypes of ADAMTS13 and vWF with CAD risk in Thais. A case-control study was performed in 197 CAD and 135 non-CAD patients. Genetic polymorphisms of ADAMTS13 (P475S, Q448E, rs2073932, P618A, A900V, S903L, rs652600, and rs4962153) and vWF (V1565L and Y1584C) along with ADAMTS13 activity, vWF antigen and vWF activity were examined in the patients. The vWF V1565L polymorphism was associated with increased ADAMTS13 activity, whereas none of ADAMTS13 polymorphisms or haplotypes was associated with its activity. Interestingly, haplotype analysis indicated that the QAGA or H4 haplotype of ADAMTS13 gene had a protective effect on CAD after adjustment for ABO blood group [odds ratio (OR) = 0.3, 95% confidence interval (CI) = 0.1, 0.6] and major CAD risk factors (OR = 0.3, 95% CI = 0.1, 0.7). However, the combination of H4 haplotype and the L allele of V1565L was not associated with increased ADAMTS13 activity when compared with the V allele. ADAMTS13 haplotype had an independent protective effect on CAD and genetic variation of vWF V1565L polymorphism modulates ADAMTS13 activity.

SiRNA-knockdown of ADAMTS-13 modulates endothelial cell angiogenesis

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA and the effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions. 15nM of ADAMTS-13 siRNA reduced HUVEC ADAMTS-13 protein levels by 90% after 24h incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Furthermore, this transfection did not affect the HUVEC endogenous protein level of ADAMTS-1, a related family member of ADAMTS-13 indicating the specificity of the siRNA. Transfection of HUVEC with 15nM of ADAMTS-13 siRNA resulted in a 21% decrease in proliferation after 24h incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24h after transfection with control siRNA, there was increased cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased protein levels of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. The protein level of vascular endothelial growth factor (VEGF), a well-known regulator of angiogenesis, was significantly decreased by 45% upon knockdown of ADAMTS-13. Moreover, activity of the AKT pathway, one of the VEGF angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through VEGF and AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.

Prognostic value of ADAMTS13 in patients with severe sepsis and septic shock.

ADAMTS13 level was evaluated as a predictor of mortality in patients with severe sepsis and septic shock, and compared with Acute Physiology and Chronic Health Evaluation II (APACHE II) scores. This prospective observational study was conducted in the Medical and Surgical Intensive Care Units of King Khalid University Hospital. Detailed clinical evaluations were performed on 84 patients (56.08±18.18 years of age) with severe sepsis and septic shock. ADAMTS13 levels were determined (three blood samples at 24 hours intervals) and APACHE II scores, hematological profiles, indices of organ hypo-perfusion, renal functions and coagulation profiles were recorded. Primary outcome was 30 days ICU mortality and secondary outcomes were its comparison with APACHE II score, length of ICU stay and use of vasopressor agents. Hypertension (53.6%) and diabetic mellitus (45.2%) were the commonest comorbidities. The median ADAMTS13 levels were 336.65, 339.35 and 313.9, respectively. ROC analysis showed maximum area under the curve for second ADAMTS13 (AUC=0.760) compared with first (AUC=0.660) and third samples (AUC=0.707) and APACHE II scores (AUC=0.662). Patients were divided into low and high ADAMTS13 groups according to the best cut-off point. Mortality was high in the low ADAMTS13 level group [OR=4.5]and was significantly associated with age, DBP, ADAMTS13, APACHE II score, DIC score and platelet count. ADAMTS13 (OR=5.3), APACHE II (OR=4.13) and DIC scores (OR=7.32) were significant risk factors for mortality. Low ADAMTS13 was associated with increased mortality in patients with severe sepsis and septic shock and was comparable to APACHE II scores for predicting mortality.

Long-Term Prevention of Congenital Thrombotic Thrombocytopenic Purpura in ADAMTS13 Knockout Mice by Sleeping Beauty Transposon-Mediated Gene Therapy.

Severe deficiency in the von Willebrand factor-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) because of mutations in the ADAMTS13 gene can lead to acute episodes of congenital thrombotic thrombocytopenic purpura (TTP), requiring prompt treatment. Current treatment consists of therapeutic or prophylactic infusions of fresh frozen plasma. However, lifelong treatment with plasma products is a stressful therapy for TTP patients. Here, we describe the use of the nonviral sleeping beauty (SB) transposon system as a gene therapeutic approach to realize lifelong expression of ADAMTS13 and subsequent protection against congenital TTP. We demonstrated that hydrodynamic tail vein injection of the SB100X system expressing murine ADAMTS13 in Adamts13-/- mice resulted in long-term expression of supraphysiological levels of transgene ADAMTS13 over a period of 25 weeks. Stably expressed ADAMTS13 efficiently removed the prothrombotic ultralarge von Willebrand factor multimers present in the circulation of Adamts13-/- mice. Moreover, mice stably expressing ADAMTS13 were protected against TTP. The treated mice did not develop severe thrombocytopenia or did organ damage occur when triggered with recombinant von Willebrand factor, and this up to 20 weeks after gene transfer. These data demonstrate the feasibility of using SB100X-mediated gene therapy to achieve sustained expression of transgene ADAMTS13 and long-term prophylaxis against TTP in Adamts13-/- mice.