Plasmin-cleaved von Willebrand factor as a biomarker for microvascular thrombosis

von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. Here, we describe the development of a variable domain of heavy-chain-only antibody (VHH)-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in patients with TTP during acute attacks of thrombotic microangiopathy but not in those in remission. Finally, we show that therapeutic plasminogen activation in a mouse model of TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion

Predictors of bleeding complications during catHeter-dirEcted thrombolysis for peripheral arterial occlusions (POCHET)

Introduction The risk of major bleeding complications in catheter directed thrombolysis (CDT) for acute limb ischemia (ALI) remains high, with reported major bleeding complication rates in up to 1 in every 10 treated patients. Fibrinogen was the only predictive marker used for bleeding complications in CDT, despite the lack of high quality evidence to support this. Therefore, recent international guidelines recommend against the use of fibrinogen during CDT. However, no alternative biomarkers exist to effectively predict CDT-related bleeding complications. The aim of the POCHET biobank is to prospectively assess the rate and etiology of bleeding complications during CDT and to provide a biobank of blood samples to investigate potential novel biomarkers to predict bleeding complications during CDT. Methods The POCHET biobank is a multicentre prospective biobank. After informed consent, all consecutive patients with lower extremity ALI eligible for CDT are included. All patients are treated according to a predefined standard operating procedure which is aligned in all participating centres. Baseline and follow-up data are collected. Prior to CDT and subsequently every six hours, venous blood samples are obtained and stored in the biobank for future analyses. The primary outcome is the occurrence of non-access related major bleeding complications, which is assessed by an independent adjudication committee. Secondary outcomes are non-major bleeding complications and other CDT related complications. Proposed biomarkers to be investigated include fibrinogen, to end the debate on its usefulness, anti-plasmin and D-Dimer. Discussion and conclusion The POCHET biobank provides contemporary data and outcomes of patients during CDT for ALI, coupled with their blood samples taken prior and during CDT. Thereby, the POCHET biobank is a real world monitor on biomarkers during CDT, supporting a broad spectrum of future research for the identification of patients at high risk for bleeding complications during CDT and to identify new biomarkers to enhance safety in CDT treatment

Platelets of patients with chronic kidney disease demonstrate deficient platelet reactivity in vitro

<p>Abstract</p> <p>Background</p> <p>In patients with chronic kidney disease studies focusing on platelet function and properties often are non-conclusive whereas only few studies use functional platelet tests. In this study we evaluated a recently developed functional flow cytometry based assay for the analysis of platelet function in chronic kidney disease.</p> <p>Methods</p> <p>Platelet reactivity was measured using flow cytometric analysis. Platelets in whole blood were triggered with different concentrations of agonists (TRAP, ADP, CRP). Platelet activation was quantified with staining for P-selectin, measuring the mean fluorescence intensity. Area under the curve and the concentration of half-maximal response were determined.</p> <p>Results</p> <p>We studied 23 patients with chronic kidney disease (9 patients with cardiorenal failure and 14 patients with end stage renal disease) and 19 healthy controls. Expression of P-selectin on the platelet surface measured as mean fluorescence intensity was significantly less in chronic kidney disease patients compared to controls after maximal stimulation with TRAP (9.7 (7.9-10.8) vs. 11.4 (9.2-12.2), P = 0.032), ADP (1.6 (1.2-2.1) vs. 2.6 (1.9-3.5), P = 0.002) and CRP (9.2 (8.5-10.8) vs. 11.5 (9.5-12.9), P = 0.004). Also the area under the curve was significantly different. There was no significant difference in half-maximal response between both groups.</p> <p>Conclusion</p> <p>In this study we found that patients with chronic kidney disease show reduced platelet reactivity in response of ADP, TRAP and CRP compared to controls. These results contribute to our understanding of the aberrant platelet function observed in patients with chronic kidney disease and emphasize the significance of using functional whole blood platelet activation assays.</p

Severe Dengue Is Associated with Consumption of von Willebrand Factor and Its Cleaving Enzyme ADAMTS-13

Severe dengue infections are characterized by thrombocytopenia, clinical bleeding and plasma leakage. Activation of the endothelium, the inner lining of blood vessels, leads to the secretion of storage granules called Weibel Palade bodies (WPBs). We demonstrated that severe dengue in Indonesian children is associated with a strong increase in plasma levels of the WPB constituents von Willebrand factor (VWF), VWF propeptide and osteoprotegerin (OPG). An increased amount of the hemostatic protein VWF was in a hyperreactive, platelet binding conformation, and this was most pronounced in the children who died. VWF levels at enrollment were lower than expected from concurrent VWF propeptide and OPG levels and VWF levels did not correlate well with markers of disease severity. Together, this suggests that VWF is being consumed during severe dengue. Circulating levels of the VWF-cleaving enzyme ADAMTS-13 were reduced. VWF is a multimeric protein and a subset of children had a decrease in large and intermediate VWF multimers at discharge. In conclusion, severe dengue is associated with exocytosis of WPBs with consumption of VWF and low ADAMTS-13 activity levels. This may contribute to the thrombocytopenia and complications of dengue

Fatal cerebral hemorrhage in a patient with thrombotic thrombocytopenic purpura with a normal platelet count during treatment with caplacizumab

Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy with a severe mortality and morbidity. Caplacizumab has recently been approved in the Netherlands as a new therapeutic option in patients with life-threatening organ failure due to aTTP. We describe the case of a 50 year old patient with aTTP who was referred to our hospital for treatment with caplacizumab. After undergoing treatment with plasmapheresis, prednisolone, rituximab and caplacizumab, her platelet count recovered and she was ready to be discharged. Unfortunately, before discharge she developed a fatal intra-cerebral hemorrhage. Fatal hemorrhage as an adverse event of caplacizumab has not been described before. Up to now there is no evidence-based treatment for caplacizumab induced heavy bleeding