ACTIVATION OF PROTHROMBIN

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This research program is aimed at understanding how thrombin is generated and how thrombin generation is regulated. Our approach to these questions comes via the convergence of four separate directions associated with 1) the physical properties of coagulation enzyme complexes, their constituents and how these complexes can assemble into efficient enzyme catalysts. 2) Studies in which multiple coagulation catalysts are mixed to attempt to duplicate the performance of the combined catalyst system such as that associated with the tissue factor pathway of thrombin expression. 3) To study this process in unadulterated minimally modified biological systems (whole blood) to evaluate the correctness of hypotheses derived from purified systems. 4) To attempt to create mathematical models which can be used to define, on a quantitative basis, the process of blood clotting and its regulation both to aid in experimental designs 1, 2, 3, and also to aid in the evaluation of the pharmacologic agents, hemostatic and thrombotic diseases. The aim of the present investigation is to understand the nature of procoagulant and anticoagulant vitamin-K dependent complexes and their regulation during the process of thrombin generation. Studies will employ physical chemistry techniques including hydrodynamics and fluorescence spectroscopy, (the latter both in solution and on surfaces) to study complexes on synthetic membranes and cells. Reactions will be followed using both synthetic and natural substrates to monitor both presteady state and steady state kinetic events. Natural and recombinant inhibitors will be used to study the regulation of procoagulant and anticoagulant processes associated with thrombin generation. We will integrate the detailed information available through studies of individual reactions with that obtained from multi-reaction center systems. The relevance of these purified systems to events occurring in whole blood will be evaluated quantitatively by our system. Conversely, the processes noted to occur in the whole blood system will direct appropriate attention in the purified system analyses. We anticipate developing a quantitative evaluation of the biologically relevant chemistry associated with the complex reactions which occur simultaneously during a blood clotting event. These data have significance in interpreting normal physiology and in developing approaches to correct the coagulation pathology associated with thrombosis and hemophilia. The techniques we develop will provide tools for the evaluation of potential pharmacological intervention in hemostatic and thrombotic disease.