Protein S K196E mutation reduces its cofactor activity for APC but not for TFPI

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.