Targeting the protein S–C4BP-β interaction: A potential therapeutic strategy for hemophilia B

Abstract

Abstract Protein S (PS) is a vitamin K-dependent, multi-domain glycoprotein composed of an N-terminal γ-carboxyglutamic acid (Gla) domain, a thrombin-sensitive region (TSR), four epidermal growth factor (EGF)-like domains, and a C-terminal sex hormone-binding globulin (SHBG)-like domain containing two laminin G-type (LG) globular domains. Traditionally recognized as a cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI), PS has recently been identified as a direct inhibitor of Factor IXa (FIXa). In addition to its anticoagulant role, PS regulates the complement system by facilitating the binding of C4b-binding protein (C4BP) to phospholipid surfaces of apoptotic cells, activated platelets, and microparticles. This interaction involves the N-terminal complement control protein (CCP) domain of C4BP-β and is believed to occur through the SHBG-like region of PS, with both LG1 and LG2 domains contributing, although the exact PS residues involved remain unclear. Approximately 60% of circulating PS is bound to C4BP, with the remaining 40% circulating freely; the anticoagulant function is primarily attributed to the free form. While most studies report that C4BP-bound PS lacks anticoagulant activity, some suggest it may retain limited direct inhibitory capacity. In this study, we investigated whether the β-chain of C4BP modulates the ability of PS to directly inhibit FIXa. Our previous work demonstrated that the LG domains of PS interact with the heparin-binding exosite of FIXa. Since the C4BP-β binding site also lies within the LG domains, we hypothesized that steric hindrance or overlapping binding interfaces may reduce PS-mediated FIXa inhibition when PS is complexed with C4BP-β. To test this, we pre-incubated equimolar amounts of PS with either C4BP or isolated C4BP-β, then assessed their effect on FIXa-mediated activation of Factor X (FX) in the presence of phosphatidylserine/phosphatidylcholine vesicles. FXa generation was quantified using chromogenic substrate S-2765 at 405 nm. As expected, 50 nM PS alone inhibited FIXa activity by ~50%. The PS–C4BP complex nearly abolished FIXa activity. However, the PS–C4BP-β complex attenuated PS function, with increasing concentrations of C4BP-β progressively restoring FIXa activity. This effect was consistent across multiple FX concentrations. These findings suggest that the C4BP-β chain interferes with the direct inhibitory action of PS on FIXa, potentially by competing for binding at the LG domains. This insight opens new avenues for developing FIXa-targeting therapeutic peptides that mimic the C4BP-β interaction with PS, offering a novel strategy for managing Hemophilia B. Future studies will focus on elucidating the structural basis of PS–C4BP interactions to further define this regulatory mechanism.