In this study, the ability of factor Xa to protect factor Va from proteolysis by activated protein C (APC) was verified. Interestingly, factor X was found to exert a similar effect with a dose-dependence identical to that of factor Xa. The effects of factor X and Xa were abrogated in the presence of protein S. To further assess the interactions of factor Va with factors Xa, X and APC, direct binding studies were performed. Factor X and Xa bound to factor Va with equal efficacy. Both proteins displaced APC from its factor Va binding site. These interactions were calcium-dependent. Although the binding of factor Xa devoid of its principal calcium binding site (the Gla-domain) to factor Va was identical to that observed using the native protein, its APC inhibitory effects were significantly reduced. These findings suggest that the Gla-domain of factor X (Xa) is pivotal in the protection of factor Va from APC.