Abstract
BACKGROUND: Protein S (PS) is an anticoagulant molecule that functions as a cofactor for activated protein C (APC) in the inactivation of activated coagulation factors Va (FVa) and VIIIa. It also serves as a cofactor for tissue factor pathway inhibitor (TFPI) in the efficient inhibition of factor Xa (FXa). The Lys(196)-to-Glu (K196E, Tokushima) mutation in the EGF-2 domain of PS is a genetic risk factor for venous thromboembolism (VTE) in the Japanese population. OBJECTIVES: To investigate the molecular basis of the thrombophilic phenotype of Japanese patients carrying the PS K196E mutation. METHODS: We expressed recombinant human PS wild-type (PS-K) and K196E-mutant (PS-E) in CHO cells, and purified them by Ni(2+)-affinity and anion exchange column chromatography. We investigated the anticoagulant functions of PS-K and PS-E by measuring APC cofactor activity, TFPI cofactor activity, affinity for the β chain of complement component C4b-binding protein (C4BP), and cleavage by thrombin. RESULTS: PS-E had approximately 40% APC cofactor activity compared with PS-K in a clotting-based assay and a FVa inactivation assay. The TFPI cofactor activity of PS-E in the FXa inactivation assay was equivalent to that of PS-K in the absence and presence of coagulation factor V. The strengths of PS-E and PS-K binding to the β chain of C4BP were comparable, and both were equally cleaved by thrombin. CONCLUSIONS: The PS K196E mutation increases the risk of VTE because of reduced APC cofactor activity but does not alter various other properties, including the TFPI cofactor activity.