THE FUNCTIONS OF PROTEIN C

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Activated protein C is a potent anticoagulant which also stimulates fibrinolysis. Activation of protein C occurs rapidly when thrombin binds to a specific endothelial cell surface receptor, thrombomodulin. Thrombomodulin accelerates thrombin catalyzed protein C activation in a Ca++ dependent reaction. The role of Ca++ in the activation of protein C will be analyzed kinetically with both protein C and protein C from which the Gla domain has been removed as the substrate. Preliminary data suggests the presence of a high affinity Ca++ binding site on protein C distinct from the Gla dependent Ca++ binding sites. This site will be studied by equilibrium dialysis and by intrinsic fluorescence changes. Thrombomodulin is normally a membrane protein. Thrombin catalyzed protein C activation is accelerated by reconstitution of thrombomodulin into membranes. The conditions for reconstitution of thrombomodulin into membranes will be determined and the specificity of the lipids studied. The kinetics of the reconstituted system will be re-evaluated to determine the nature of the lipid enhancement. The thrombomodulin concentration on the endothelial cell surface is not known. A monoclonal antibody to thrombomodulin will be produced and used to determine the number of thrombomodulin molecules/endothelial cell. From kinetic studies it will be possible to determine if the endothelial cell provides unique properties which cannot be duplicated by synthetic systems. Human thrombomodulin will be isolated and characterized as done previously for rabbit thrombomodulin. Kinetic studies on human protein C, human thrombomodulin and human endothelial cells will be performed. Activated protein C mediated anticoagulant activity is not completely characterized. Although activated protein C inhibits factor Va the rate of inactivation is too slow to account for the anticoagulant effect. Protein S stimulates factor Va inactivation, but the extent of stimulation in vitro does not account for the plasma stimulation. The influence of low levels of activated protein C on the time course of both prothrombin and factor V activation in plasma and in purified systems will be monitored to determine if protein C alone or with protein S influences the lag before prothrombin activation occurs or whether it influences the rate and/or extent of prothrombin consumption. The ability of platelets to support activated protein C anticoagulant activity will also be investigated.