This research proposal is designed to extend the biochemical and clinical knowledge of human protein C and its plasma inhibitor. Protein C, a vitamin K-dependent zymogen which exhibits both anticoagulant and profibrinolytic activity is part of an antithrombotic regulatory system. Activated protein C (APC) itself is regulated by a plasma protein inhibitor. The major specific aims of this proposal are : 1) isolation and characterization of the plasma inhibitor of APC; 2) characterization of the mechanism and kinetics of the inhibition of APC; 3) establishment of assays for protein C, APC, the inhibitor of APC and the APC-inhibitor complex; and 4) investigation of the role of protein C and its inhibitor in plasma of patients with hereditary and acquired thromboembolic complications. The inhibitor of APC will be isolated using a variety of separation techniques and characterized by multiple chemical, physical and immunological techniques. These characteristics will serve as baseline data for the interaction studies and for comparison to other protease inhibitors. An in-depth study of the mechanism of action (stoichiometric vs. catalytic) will be performed. The basic kinetics of inactivation will be determined using pseudo-first order techniques, and more complex kinetics (heparin potentiation) will be described, if necessary. The effects of ions, pH and other factors will also be tested. Functional and immunological tests for all forms of protein C and its inhibitor will be developed and validated for plasma; and the normal ranges of each test will be determined. Levels of protein C, its inhibitor and other forms will be determined in complete family studies of patients with hereditary recurrent thromboembolic disease or combined Factor V/VIII deficiency. The levels of protein C related molecules will also be determined in serial samples of patients with DIC or acquired thromboembolic complications. This information will provide data on the fluctuations of the protein C system in these disease conditions. The proposed studies should provide fundamental biochemical and clinical information about the control mechanisms of the protein C regulatory system. This new knowledge may help to establish the usefulness of protein C assays in the diagnosis and treatment of DIC and thromboembolic disorders.
