The hemostasis system is subjected to exacting regulation to meet on one hand the demands of homeostasis while on the other to avert decompensation towards thrombosis which, in context of arterial disease, is the leading cause of morbidity and mortality in western cultures. The proposed research is aimed broadly at mechanisms that limit the activity of the hemostasis system to sites of injury, with a view of the long term goal of enabling strategies for detection, suppression and ultimately prevention of thrombosis. The focus of the proposal is on mechanisms of regulation by and of the proteases that comprise the hemostasis system, particularly the control of protease specificity by regulatory subunits, by substrate structure and by inhibitors. The focal protease is thrombin, which has a plastic specificity determined by a variety of plasma and cellular effectors including heparin and other polyanions, acidic peptides, a regulatory subunit (thrombomodulin) and inhibitors. A major aim of the proposal elucidation of tertiary structural details on thrombin that are involved in interactions with effectors. The major experimental approaches is measurement of the influence of effector binding on the rate constants of reductive methylation of every lysyl residue in thrombin. In parallel, the capacity of each effector to compete with each other will be evaluated. The goal is a tertiary/quaternary structure for each thrombin-effector dimer. A related but experimentally distinct aim is the nature of the platelet membrane protein that interacts with thrombin to initiate platelet activation. The working hypothesis is that hydrolysis of a membrane protein is the initiating event. The protein will be characterized by the relative susceptibility of platelet activation to thrombins or thrombin derivatives having a broad and non-parallel distribution of specific activities toward known thrombin substrates. The thrombins will be purified from plasmas of mammals, birds, reptiles and fish, and a limited group of chemical and genetic mutants will be used. The goal is characterization of the protein sufficiently to enable its physical identification and isolation. Many of the technical developments will be capitalized on to develop a thorough, conventional enzymology of the cognate protease, factor IXa, which is likewise regulated but is poorly characterized as an enzyme. The goal is an understanding of factor IXa at a level comparable to that of thrombin. Included in the goal is the regulation of factor IXa and its substrate/product factor X/Xa by the microvascular endothelium. The latter goal will be addressed in perfused rat hearts.
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