The theme of this project is to understand how complexes of proteins within the blood clotting cascade assemble on membrane surfaces and how this assembly process regulates enzymatic activity. The proposed studies focus on assembly of the cell-surface complex that triggers blood clotting, consisting of the regulatory protein, tissue factor, and its cognate serine protease, factor Vila;and investigating the mechanism of action of novel regulators of blood clotting that we have recently discovered. Specifically, we will investigate the following research questions: (1) How does membrane topography contribute to tissue factor function? These studies will examine the consequences of altering the membrane topography of tissue factor on the proteolytic activity of the tissue factor-factor Vila complex. Included is an investigation of a novel way of attaching the extracellular domain of tissue factor (sTF) to membrane surfaces employing oligohistidine-tagged sTF and metal ion-chelating lipids. (2) How does polyphosphate modulate the blood clotting cascade? We discovered that polyphosphate, an ancient molecule that is abundant in many microorganisms, and newly discovered in dense granules of human platelets, is a potent modulator of blood clotting and fibrinolysis. We will examine: (a) platelet secretion of polyphosphate, including studies comparing normal platelets and platelets from patients with Hermansky-Pudlak syndrome;(b) the ability of polyphosphate to stimulate the contact pathway of blood clotting;(c) the mechanism by which polyphosphate completely abrogates the anticoagulant function of tissue factor pathway inhibitor (TFPI);(d) how polyphosphate modulates thrombin function;and (e) how expression of polyphosphate in bacteria (Escherichia coli and Salmonella typhimurium) modulates the blood clotting and fibrinolytic cascades. These studies are aimed at understanding how newly discovered modulators of the blood clotting system contribute to normal hemostasis and to the activation of the blood clotting system in human disease.
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