Identification of the Protein S Binding Site in the Factor IXa Serine Protease Domain Presents Therapeutic Possibilities

Protein S (PS) has been shown to inhibit Factor IXa (FIXa); however, its binding sites on FIXa have not been fully elucidated. By structural analysis of FIXa in conjunction with previous studies of FIXa's ability to bind heparin, an FIXa inhibitor, we identified that multiple residues that make up the heparin binding exosite, including Lys¹²⁶, Asn¹²⁹, Lys¹³², Arg¹⁵⁰, Arg¹⁶⁵, and Arg¹⁷⁰ (chymotrypsin numbering), might contribute to binding PS; these residues were substituted with either Ala or Leu in separate FIXa mutants. Factor Xa (FXa) generation assays along with fluorescence analyses provided both the catalytic efficiencies and dissociation constants for the FIXa mutants under conditions of varied PS. Furthermore, anisotropic measurements of the fluorescent DEGR-FIXa's binding interaction with PS, when titrated against each of the FIXa mutants, supported the data found in the previous assays. These studies suggested that multiple residues, which are critical for the binding of heparin, such as Lys¹²⁶, Lys¹³², Arg¹⁶⁵, and Arg¹⁷⁰, are also required in binding PS. To additionally verify this data, further ex vivo studies, including co-immunoprecipitation and flow cytometry of PS and FIXa are underway. Although previous studies have shown that the first EGF-like domain of FIXa is required to bind PS, these novel results have not only localized the main inhibitory site of PS on FIXa, but also provided enough information to propose a binding mechanism, which may rely on the reciprocal association between the head groups of PS and FIXa with the other's EGF-like domains, forming a bridging structure upon the cell membrane. Further studies are required to verify this hypothesis, but when clarified, it could provide immense benefits in the production of therapeutics for thrombosis.