Since plasma protein S serves an anticoagulant function by mechanisms which are not completely understood, its possible interaction with Factor Va was investigated. Human protein S bound to immobilized human Factor Va in a calcium-dependent, saturable, and reversible manner and Factor Va bound similarly to immobilized protein S. Binding of protein S to immobilized Factor V was greatly enhanced by pretreatment of the surface-bound Factor V with increasing doses of thrombin up to 1 unit/ml. Binding of protein S to Factor Va was also demonstrated in fluid phase with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized protein S, and this binding was reversed by a 17-fold molar excess of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinase activity in a reaction mixture of purified clotting factors was inhibited by protein S and exhibited a pattern of mixed inhibition. The concentration of protein S needed for 50% inhibition of the prothrombinase activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phospholipids was about 16 nM. Since not all protein S preparations exhibited this degree of prothrombinase inhibitory activity, extensive control experiments were performed to verify that the inhibitory activity was associated with protein S during immunoaffinity chromatography and was not caused by traces of activated protein C in the protein S preparations. These data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.