Identity of Plasma-activated Factor X Inhibitor with Antithrombin III and Heparin Cofactor

Abstract

Abstract Antithrombin III activity cochromatographed with the activity of the inhibitor to activated Factor X during the purification of the inhibitor on Sephadex G-200, DEAE-Sephadex, and DEAE-cellulose columns. Both activities also comigrated on preparative polyacrylamide gel disc electrophoresis. When a fraction of activated Factor X inhibitor was incubated at 56°, at pH 7.5, a simultaneous loss of antithrombin III and activated Factor X inhibitor activities resulted. A complete neutralization of the antithrombin III activity of the activated Factor X inhibitor fraction with thrombin rendered the inhibitor incapable of subsequently inhibiting activated Factor X and vice versa. When the inhibitor was chromatographed on a Sephadex G-200 column, antithrombin III, heparin cofactor, and activated Factor X inhibitor activities cochromatographed with the 280 mµ absorbing material in the activated Factor X inhibitor sample applied. To demonstrate further the identity of these three anticoagulant activities, a series of gel filtration experiments on a single column of Sephadex G-200 was performed in which fractions of activated Factor X inhibitor alone, heparin alone, and heparin mixed with the inhibitor were separately chromatographed in the presence of 0.01 m, 0.10 m, 0.20 m,and 0.30 m NaCl in 0.02 m Tris-maleate, pH 7.2, at 26°. The relative elution volume, Ve/V0 of activated Factor X inhibitor alone in 0.01 m NaCl was 1.83, and in 0.10 m to 0.30m NaCl was 1.77. Heparin behaved as a heterogenous substance with molecular weights ranging from 40,000 to greater than 200,000, at the salt concentrations studied. When a mixture of activated Factor X inhibitor and heparin was chromatographed, the two components eluted as a complex. A single broad heparin cofactor peak cochromatographed with the activated Factor X inhibitor protein and the heparin when high ionic strength eluent was employed. The fractions demonstrated antithrombin III activity when the heparin in the heparin cofactor peak fractions was neutralized with protamine sulfate. The plasma-activated Factor X inhibitor was shown to be unrelated to a hepatic anticoagulant that had been claimed to be a specific inhibitor of activated Factor X. It is concluded, therefore, that the biological activities variously termed activated Factor X inhibitor, antithrombin III, and heparin cofactor activities all belong to a single blood proteinase inhibitor with broad specificity. Together with other data presented, it is suggested that the key function of activated Factor X inhibitor as a natural anticoagulant may be primarily concerned with regulating hemostatic balance through neutralization of activated Factor X, a reaction profoundly enhanced by traces of heparin. It is for these reasons we propose that this low molecular weight α2-globulin inhibitor be termed activated Factor X inhibitor.