Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.