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

Masahiro Takeyama, Shoko Furukawa, Kana Sasai, Kaoru Horiuchi, Keiji Nogami

Biochimica et biophysica acta. General subjects 2023 Aug

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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. To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798. 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) (IC50; 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 KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax 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. The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site. Copyright © 2023 Elsevier B.V. All rights reserved.

Citation

Masahiro Takeyama, Shoko Furukawa, Kana Sasai, Kaoru Horiuchi, Keiji Nogami. Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex. Biochimica et biophysica acta. General subjects. 2023 Aug;1867(8):130381