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

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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.