This research grant application is designed to provide basic knowledge about a new human plasma protein, protein C (the precursor of autoprothrombin II-A), that remarkably exhibits both anticoagulant activity and profibrinolytic activity. Protein C is a plasma serine protease zymogen that is activated by limited proteolysis by thrombin. The major objectives of this application include the isolation and the chemical and physical characterization of protein C and activated protein C using multiple biochemical techniques. The activation of protein C by thrombin will be studied in detail. The mechanism of action of activated protein C as a anticoagulant will be defined. In these studies the potential influence of activated protein C on coagulation Factors V, VIII, IX, X, XI, and XII, prekallikrein and high MW kininogen will be explored. The mechanism of action of activated protein C as a profibrinolytic agent will be studied for these studies, potential interactions between protein C and platelets, cultured endothelial cells, and subendothelium structural elements will be probed. The potential influence of activated protein C on purified plasminogen and on alpha 2 antiplasmin will be defined. Since plasma can inhibit activated protein C, experiments will be performed to identify the mechanism of inhibition of this molecule by other plasma components. The importance of proteolytic inactivation and of plasma protease inhibitors in this reaction will be assessed. Antithrombotic therapy currently may involve antiplatelet therapy (drugs), anticoagulant therapy (heparin or coumarins) or fibrinolytic therapy (urokinase or streptokinase). Currently in the U. S. A., there is no antithrombic therapy involving plasma proteins. The studies proposed here will be most useful for assessing the potential usefulness of protein C as a new antithrombotic agent that may supplement or replace current therapeutic agents.
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