Laminin G domains define a critical interface for protein S-mediated factor IXa inhibition

Hemostasis is maintained through a delicate balance between procoagulant and anticoagulant mechanisms. Protein S (PS), a multidomain, vitamin K-dependent glycoprotein, contributes to this balance not only as a cofactor for activated protein C and tissue factor pathway inhibitor but also by directly inhibiting activated factor IX (FIXa). However, the structural determinants underlying FIXa inhibition remain unclear.

This study aimed to (1) identify the FIXa binding interface on PS and (2) investigate the role of its laminin G (LG) domains in mediating FIXa binding and inhibition.

Molecular docking was used to predict the FIXa binding interface on PS. Fluorescence-based binding assays were performed to determine binding affinities, and functional coagulation assays were conducted to measure inhibitory constants. Finally, site-directed mutagenesis was carried out to generate specific PS mutants.

Molecular docking and in vitro binding assays demonstrated that both the LG1 and LG2 domains interact with FIXa. Quantitative fluorescence-based binding analyses revealed that the LG1+2 tandem domains exhibited the highest affinity for FIXa (K_d ≈ 52.15 nM). Functional coagulation assays showed that LG1+2 effectively blocked FIXa-mediated factor X activation and suppressed thrombin generation. Furthermore, site-directed mutagenesis confirmed that residues E435 and E437 within the LG domains are critical for FIXa binding and inhibition.

These findings identify the LG domains of PS as essential structural elements for direct FIXa inhibition, independent of its cofactor function. By elucidating this domain-specific mechanism, our work provides a structural framework for the rational design of selective FIXa inhibitors and FIXa-binding peptides as novel antithrombotic strategies.