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

致命的动脉血栓形成，如心肌梗塞或中风，被认为是由止血机制触发的，而止血机制最初是人类防御所必需的。止血是通过血小板黏附/聚集和凝血机制的协同作用而建立的。有趣的是，在血小板黏附/聚集中起重要作用的von Willebrand因子(VWF)在血浆中与凝血因子VIII形成二元复合体，凝血因子VIII是主要的凝血因子。因此，本研究旨在阐明凝血因子VIII/VWF这一关键分子复合体在全血条件下的激活或失活机制，并建立基于凝血因子VIII/VWF复合体功能调节的新型抗血栓策略。因此，Arg-336在第VIII因子…中的切割更多的分子被认为是纤溶酶催化的因子失活的核心机制。接下来，关于VWF的调节，我们研究了全血条件下ADAMTS13活性的分子机制，适当的VWF功能是正常止血所必需的，但VWF的过度功能与动脉血栓形成有关。ADAMTS13被认为在体内通过减少直接代表VWF血栓形成活性的多聚体大小来调节VWF的功能，但其确切的作用机制尚不清楚。采用体外灌注室系统，研究了ADAMTS13在全血流条件下对胶原表面血小板血栓形成过程的影响。功能阻断的抗ADAMTS13抗体的抑制研究，以及能够特异性反映ADAMTS13切割VWF活性的AND-VWF单抗对血栓的免疫染色，证明了ADAMTS13的剪切率依赖作用在正在进行的血栓形成过程中限制血栓生长的视觉证据。我们的结果揭示了在高切变率条件下微妙地防止动脉闭塞的壁血栓形成的调节机制。较少

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