Biological Properties of the Naturally Occurring Plasma Inhibitor to Activated Factor X

Abstract Highly purified fractions of the plasma inhibitor of activated Factor X were found to inhibit purified thrombin, but had no effect on activated Factors XI and XII or on nonactivated Factor X. The inhibitor was greater than 30 times more effective towards activated Factor X than thrombin, on a weight basis. The inhibition of the biological activity of both enzymes by activated Factor X inhibitor was progressive and irreversible. Irreversible inhibition of activated Factor X by its inhibitor was also demonstrated by employing a purified fraction of goat antibody developed against the inhibitor. Both the coagulant and esterase activities of activated Factor X and thrombin were neutralized by activated Factor X inhibitor. In the presence of optimal amounts of heparin and activated Factor X inhibitor, the destruction of the coagulant activity of activated Factor X and the blockade of the thrombin coagulant activity occurred. The esterase activity of activated Factor X was similarly affected in the presence of heparin, but that of thrombin was unaffected. In the presence of heparin, the complete and irreversible inactivation of the biological activity of activated Factor X by its inhibitor remained unchanged when the heparin was subsequently neutralized with protamine sulfate. In contrast, the thrombin clotting activity blocked by activated Factor X inhibitor in the presence of heparin was quantitatively recovered when the heparin was neutralized by protamine sulfate. The complete recovery of biologically active thrombin was dependent upon the pH of the test system and the ratio of activated Factor X inhibitor to heparin. These data indicate that the efficiency of activated Factor X inhibitor as an anticoagulant during normal blood coagulation (an action profoundly enhanced by heparin) may depend more on its preventing any generated activated Factor X from activating prothrombin than it may on preventing thrombin from attacking fibrinogen.