Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S-deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, ␥-carboxylated and bound phospholipids with an apparent dissociation constant (Kd app ) similar to that of wildtype (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC. (Blood. 2010;115(23): 4878-4885) IntroductionProtein S, a vitamin K-dependent plasma anticoagulant protein, functions as an enhancing cofactor to activated protein C (APC) in the inactivation of activated factors V (FVa) and VIII (FVIIIa). 1 Protein S also has an APC-independent activity that has recently been attributed to its ability to enhance tissue factor pathway inhibitor (TFPI). 2,3 Protein S has an important role in vivo, as is shown clinically by infants with complete deficiency, who suffer purpura fulminans, and by heterozygous carriers of PROS1 gene deletions and point mutations, who are at enhanced risk of venous thromboembolism. 4,5 The importance of protein S has also been demonstrated in murine knockouts, which fail to survive development. 6,7 Protein S binds phospholipids and helps to localize APC to the membrane surface, in proximity to FVa and FVIIIa. In doing so, rather than the preferential cleavage at Arg506 of FVa by APC, Arg306 is then favored, with cleavage enhanced approximately 20-fold. 8 Although this effect has generally been accepted to be due to protein S repositioning of the active site of APC, 9 this has recently been questioned. 10 Cleavage at Arg506 and Arg306 fully inactivates FVa, thereby efficiently down-regulating coagulation. 11 APC also inactivates FVIIIa by cleaving first at Arg336 and thereafter at Arg562. In the presence of protein S, it has been shown that the efficiency of cleavage after Arg336 by APC is enhanced approximately 3-fold. 12 Protein S is a 635-amino acid glycoprotein, circulating at a plasma concentration of approximately 350nM. It is composed of an N-terminal Gla domain (amino acids 1-45), a thrombinsensitive region (TSR; amino acids 46-75)...