Mechanism of Procoagulant Activity Appearing on the Surface of Activated Platelet and New Development of Anttithrombotic Therapy by Its Inhibition.

动脉粥样硬化破裂部位血小板聚集后形成的纤维蛋白在动脉血栓形成(如急性心肌梗死)的发病中起重要作用。我们开发了一种新的体外实验系统，展示了动脉血流条件下固定化胶原表面的血小板形成血栓的促凝活性，该系统由体外流动腔系统和表观荧光显微镜组成。将含有不同浓度阿加曲班特异性抗凝血酶制剂的人血标本以1,500 S的壁剪切率灌流到固定化的I型胶原上。用AlexFluor595偶联的抗纤维蛋白单体单抗，在活化的血小板表面形成纤维蛋白单体，使其荧光为红色。我们已经证明，被认为是抗凝剂的特异性凝血酶抑制剂可以通过阻断激活的血小板表面产生的凝血酶的功能来阻止血小板血栓的三维生长。我们还发现，另一种凝血酶抑制剂Hirulog可以不可逆地阻断凝血酶的功能，更有效地阻止血小板凝血酶的三维生长。我们已经证明了血小板血栓的形成与凝血剂的激活密切相关。级联，提示血小板衍生的促凝血活性可能成为抗血栓治疗的新靶点。

Fibrin formation subsequent to the accumulation of platelet at site of disrupted atheroma play an-important role in the onset of arterial thrombosis such as acute myocardial infarction. We have developed a new ex vivo assay system, demonstrating procoagulant activity appearing on the surface of platelet formatting thrombi on the surface of immobilized collagen under arterial blood flow condition, which was composed from ex vivo flow chamber system as well as an epi-fluorescent vidio-microscopy. Human blood samples containing various concentrations of specific antithrombin agent of Argatroban were perfused on the immobilized type I collagen at a wall shear rate of 1,500 s^<-1>. Platelet, which was rendered fluorescent by addition of mepacrine, was fluorescinated as green, while fibrin monomer formed on the surface of activated platelets, which was fluorescent as red with the use of AlexFluor595 conjugated anti-fibrin monomer monoclonal antibody. We have shown that specific thrombin inhibitor, which has been considered as anticoagulant, could prevent the three-dimensional growth of platelet thrombi by blocking the function of thrombin generated on the surface of activated platelets. We have also shown that another thrombin inhibitor hirulog, which block the function of thrombin irreversibly, more efficiently prevented the three-dimensional growth of platelet thrombin. We have demonstrated the close relationship between platelet thrombus formation and the activation of coagulant. cascade, suggesting the platelet-derived procoagulant activity as a new target of antithrombotic treatment.