INHIBITION OF HUMAN FACTOR VIIA-TISSUE FACTOR BY ANTITHROMBIN III-HEPARIN IS ENHANCED BY FACTOR-X ON A HUMAN BLADDER-CARCINOMA CELL-LINE

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tis sue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not i nvolved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gla -domain in this reaction, Antibody experiments revealed that this effe ct was not due to trace levels of a tissue factor pathway inhibitor co ntaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of hepar in resulted in a factor VIIa-tissue factor neutralization rate essenti ally identical to that observed for antithrombin III alone. Plasma lev els of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pr onounced as that of factor X. Other vitamin K-dependent plasma protein s including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of m ixed phospholipids. Our collective findings suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin mole cule resulting in a transient ternary complex of antithrombin III-hepa rin-factor X that represents the anticoagulant species, Factor X conce ivably guides this putative complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue facto r complex and facilitates rapid neutralization of factor VIIa. Our fin dings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than prev iously recognized.