Protein S is a vitamin K-dependent non-enzymatic coagulation factor involved in the regulation of activated protein C (APC). In this paper we report an APC-independent anticoagulant function of protein S. We observed an inhibition of prothrombinase activity on endothelial cells and platelets which was half-maximal at physiological concentrations of free protein S in plasma. On endothelial cells, thrombin-cleaved protein S (PSt) as well as protein S in complex with C4b-binding protein (C4BP) inhibited prothrombinase activity to the same extent as protein S did. In solid-phase binding assays, direct binding of protein S and PSt to factor V and factor Va were observed. Protein S-C4BP complex did not bind to factor V or factor Va, implicating that the factor V(a) binding site on protein S is lost by the interaction with C4BP. A direct inhibition of factor Xa activity by protein S was also observed. Incubation of factor Xa with protein S revealed a noncompetitive inhibition of factor Xa by protein S with a Ki of (4.9 +/- 0.8) x 10(-7) M. Both protein S and protein S-C4BP complex were able to inhibit factor Xa to the same extent, whereas PSt had lost its inhibitory activity. This suggests that the conformational change induced by cleavage of the amino-terminal thrombin-sensitive loop results in a loss of a factor Xa binding site on protein S. The inhibitory effect of protein S involves interactions with both factor Va and factor Xa. The interaction of protein S with factor Xa is influenced by cleavage of the thrombin-sensitive loop, whereas C4BP blocks the interaction of protein S with factor Va. Inhibition of the prothrombinase complex by either forms of protein S might be an important mechanism in regulating thrombin generation in blood coagulation. The importance of the APC-independent anticoagulant action of protein S was emphasized by experiments in which the addition of protein S to normal plasma induced a prolongation of clotting time in a dilute activated partial thromboplastin time (dAPTT) assay. Furthermore, inhibition of endogenous protein S by the addition of monoclonal antibodies against protein S to normal plasma, induced a shortening of the clotting time in a dAPTT assay.