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

Document Type

Article

Publication Date

1-6-2026

Publication Title

Journal of Thrombosis and Haemostasis

Abstract

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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 ≈ 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. CONCLUSION: 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.

PubMed ID

41506583

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