Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

Background: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa.

Aim: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798.

Methods And Results: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC; 19.2 and 42.9 μM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater K compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the K by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the K was increased by 3.4-fold and the V was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction.

Conclusion: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.