The proposed investigation of molecular mechanisms involved in the regulation of human blood coagulation by factor Va forms the basis for development of a new, independent research program by the candidate. The level of blood coagulation factor Va in vivo is controlled by two opposing proteolytic reaction processes which are essential to normal hemostasis, activation of factor V by thrombin and inactivation of factor Va by activated protein C. Factor Va appears to participate in regulating its own concentration by acting as a cofactor to enhance the rate of protein C activation as well as that of prothrombin activation. A regulatory binding interaction of thrombin with factor Va has been implicated in this process which may represent a critical point in determining the balance between procoagulant and anticoagulant systems. The goal of the proposed studies is to quantitate and characterize thrombin-factor Va interactions and to elucidate mechanisms by which factor Va modulates thrombin reactions. Reaction mechanism hypotheses for the participation of thrombin-factor Va interactions in protein C and prothrombin activation will be evaluated by the use of fluorescence techniques to characterize binding interactions among the proteins and the assembly of phospholipid membrane surface-bound complexes, in conjunction with kinetic studies of the reactions. These studies will seek to define the contributions of specific protein-protein interactions, protein-membrane interactions and conformational changes to the rate enhancements. The resulting quantitative description of the poorly understood but potentially significant regulation of thrombin by human factor Va will provide the foundation for evaluating its role in normal hemostasis and in thrombotic disease. The proposed studies reflect a continuing interest of the candidate in applying physical biochemical approaches to investigate molecular mechanisms of vascular disease.
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