Novel antithrombotic strategy based on the functional regulation of factor VIII/VWF complex

致命的动脉血栓形成，如心肌梗死或中风，被认为是由止血机制引发的，而止血机制最初是人体防御所必需的。止血是血小板粘附/聚集和血液凝固机制共同作用的结果。有趣的是，在血小板粘附/聚集中起重要作用的血管性血友病因子（VWF）在血浆中与凝血因子VIII形成二元复合物，后者是主要的凝血因子。因此，本研究的目的是阐明全血条件下这一关键分子复合物（因子VIII/VWF）的激活或失活机制，并建立基于因子VIII/VWF复合物功能调控的新型抗血栓策略。关于因子VIII的调节，我们研究了纤溶因子纤溶酶对因子VIII的激活/失活。因此，Arg-336在因子VIII…More分子内的裂解被认为是纤溶酶催化因子VIII失活的主要机制。因此，我们认为，在凝血反应和纤溶活性中，纤溶蛋白通过直接的蛋白水解反应发挥调节作用。接下来，关于VWF的调控，我们研究了全血条件下ADAMTS13活性的分子机制，VWF的正常功能对正常止血至关重要，但VWF的过度功能与动脉血栓形成有关。ADAMTS13被认为通过减少直接代表VWF血栓形成活性的多聚体大小来调节体内VWF功能，但确切的作用机制仍有待阐明。利用体外灌注室系统，我们研究了全血流动条件下ADAMTS13在胶原表面血小板血栓形成过程中的活性。功能阻断抗ADAMTS13抗体的抑制研究，结合能特异性反映ADAMTS13切割vwf活性的and-VWF单克隆抗体对血栓的免疫染色，证明了ADAMTS13剪切速率依赖的作用，在正在进行的血栓生成过程中限制了血栓的生长。我们的研究结果揭示了在高剪切速率条件下壁画血栓形成的调节机制，以精确地防止动脉闭塞。少

Fatal arterial thrombosis such as myocardial infarction or stroke is assumed to be triggered by hemostatic mechanisms which are originally essential for human defense. Hemostasis is established by the concerted functions of platelet adhesion/aggregation and blood coagulation mechanisms. Interestingly, von Willebrand factor (VWF) that plays a paramount role in platelet adhesion/aggregation is known to form a binary complex in plasma with the coagulation factor VIII which is the major blood coagulation factor. Thus, the purpose of the present study is to clarify the activation or inactivation mechanisms of this crucial molecular complex (factor VIII/VWF) under whole blood flow conditions, and to establish the novel antithrombotic strategy based on the functional regulation of factor VIII/VWF complex.With regard to the factor VIII regulation, we have studied the activation/inactivation of factor VIII by a fibrinolytic factor plasmin. As a result, the cleavage of Arg-336 within factor VIII … More molecule was thought to be a central mechanism of plasmin-catalyzed factor VIII inactivation. Thus, we suggest the presence of a regulatory role of plasmin through direct proteolitic reaction in the coagulation reaction as well as fibrinolytic activity.Next, with regard to the VWF regulation, we have studied the molecular mechanisms of ADAMTS13 activity under whole blood flow conditions, The proper function of VWF is essential for normal hemostasis, but the excessive function of VWF links to arterial thrombosis. ADAMTS13 is assumed to regulate the VWF function in vivo by reducing the multimer size that directly represents the thrombogenic activity of VWF, while the precise action mechanisms remain to be clarified. Using an in vitro perfusion chamber system, we have studied the ADAMTS13 activity on the process of platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a function-blocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an and-VWF monoclonal antibody that can specifically reflect the VWF-cleaving activity of ADAMTS13, demonstrated visual evidence for a shear-rate dependent action of ADAMTS13 that limits thrombus growth at the on-going thrombus generation process. Our results revealed a regulating mechanism on mural thrombogenesis to exquisitely prevent arterial occlusion under high shear rate conditions. Less