Identification of a protein S-binding site in the C2 domain of factor VIII light chain

We demonstrated that protein S (PS) directly impairs the intrinsic tenase complex, independent of activated protein C (APC), by competitively inhibiting the binding of factor (F)IXa to the A2 domain (residues 488-490) and light chain (LCh) of FVIIIa.

This study investigated the PS-binding sites within the FVIII-LCh subunit.

Binding interactions between PS and the FVIII-LCh were characterized using ELISA, a synthetic C2-domain peptide (residues 2228-2240), and surface plasmon resonance. The interacting residue was examined using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride-mediated crosslinking and N-terminal sequencing. The functional role of the identified site (K2239) was evaluated using recombinant FVIII mutants and FXa generation assays assessing sensitivity to PS-mediated and APC/PS-mediated inhibition.

PS bound to the FVIII-C2 domain and competitively inhibited FIXa binding to the LCh. Using a synthetic peptide (2228-2240) and crosslinking, lysine 2239 (K2239) was identified as a residue contributing to PS interaction. A recombinant FVIII-K2239A mutant showed reduced binding affinity to PS (K _d, 6.7 vs 3.5 nM) and impaired PS-mediated inhibition of FXa generation. This effect was additive with mutations in the previously identified A2-domain site (S488A/R489A/R490A). Importantly, while the A2 site contributed to APC/PS-dependent inactivation, the contribution of K2239 to this process appeared limited, suggesting a more prominent role in the APC-independent pathway.

Factor VIII residues 488 to 490 and 2239 participate in APC-independent interaction with PS, while residues 488 to 490 also contribute to the APC/PS-dependent inactivation of FVIIIa.