Identification of Amino Acid Residues in Protein S LG Domains Responsible for Binding to and Inhibiting Factor IXa

Protein S (PS) is a key negative regulator of blood coagulation. Protein S deficiency and dysfunction cause life-threatening thrombotic conditions such as neonatal purpura fulminans, deep vein thrombosis, arterial thrombosis, and pulmonary embolism. Protein S has long been described as a cofactor for both Activated Protein C and Tissue Factor Pathway Inhibitor. However, we discovered that PS has an important regulatory function in the coagulation cascade, namely, direct inhibition of activated Factor IX (FIXa). This function is performed by binding between the laminin G (LG) domains of PS and the heparin-binding exosite of FIXa.

Our goal in this study was to identify the amino acid residues of PS involved in binding FIXa. In silico interaction studies predicted candidate FIXa interaction sites within PS LG domains. These sites were mutated to obtain single amino acid substitutions D345A, T372A, S394A, E435A, and E437A and a double mutant E453A/E437A, all successfully expressed in E.coli. The purity of these proteins was confirmed by SDS-PAGE followed by silver staining and immunoblotting. Using fluorescence-based interaction methods, we identified the amino acid residues that are vital for PS-FIXa interaction. The double mutant E453A/E437A had the lowest binding affinity of the mutants. The mutants were further analyzed by in vitro biochemical assays to measure their ability to inhibit FIXa's activity of converting FX to activated FXa. Interestingly, both the E453A/E437A double mutant and the single mutants E453A and E437A showed minimal inhibition of FIXa. These data suggested that the glutamic acid (E) residues at positions 453 and 437 of PS are important both for binding FIXa and for inhibiting FIXa activity.

In summary, we identified two amino acid residues in the PS LG domain required for binding FIXa and inhibition of FIXa enzymatic activity. Our results explain the structure-function relation between PS and FIXa in hemostasis. Further measurement of effects these mutants in vivo will provide a comprehensive understanding of the PS function in the coagulation pathway and potentially identify targets for therapeutic interventions to manage coagulation disorders.

No relevant conflicts of interest to declare.