Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme

Human protein C was purified, and the mechanism of action of activated protein C as an anticoagulant in plasma was studied. Protein C was purified from commercial factor IX concentrate by DEAE-Sephadex and dextran sulfate-Sepharose chromatography and preparative Polyacrylamide gel electrophoresis. Purified protein C appeared homogenous at 62,000 mol wt on nonreduced SDS-polyacrylamide gels and, following reduction, protein C gave two polypeptide chains of 40,000 and 22,000 mol wt. Protein C was activated by immobilized trypsin or thrombin or by soluble thrombin. Activated protein C markedly prolonged the prothrombin time and the activated partial thromboplastin time of normal plasma, but had no effect on the thrombin time. Activated protein C exhibited amidolytic enzyme activity. DFP inhibited both the amidolytic and anticoagulant activities. Activated protein C was added to human plasma in the presence or absence of phospholipid or calcium. This mixture was incubated for 3 min at 37°C, diluted with EDTA, and the remaining clotting activity of each of the coagulation factors was determined using appropriate deficient plasmas. Activated protein C inactivated >80% of factor V and factor VIII under these conditions, whereas factors I, II, X, VII. IX, XI, and XII, prekallikrein, and high molecular weight kininogen were not affected. Both phospholipid and calcium were required for potent inactivation of factors V and VIII in plasma. When partially purified factor VIII was activated by thrombin, it was 30 times more susceptible to inactivation than nonactivated factor VIII. These results suggest that by inactivating both factor V and factor VIII, the enzymatically active form of protein C may serve as a major physiologic regulator of cofactors of coagulation during hemostasis and thrombosis.