Journal of thrombosis and haemostasis : JTH · 2025
Defining the critical roles of factor VIII residues 1900 to 1908 in factor VIIIa-factor IXa complex formation and factor VIIIa activity.
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Lv Haixia, Ming Zhe, Wang Dongmei, Jin Mei, Li Wenqiang, Wang Wensheng
Аннотация
BACKGROUND: Factor (F)VIIIa acts as a cofactor with FIXa on phospholipid surfaces to convert FX to FXa. Region 1900 to 1908 is a known FVIIIa antibody epitope, with mutations in this region linked to hemophilia A. However, its role in FXase activity is unclear. This study investigates how individual residues within 1900 and 1908 affect interprotein affinity and cofactor activity.
OBJECTIVES: This study investigated how individual residues within region 1900 to 1908 contribute to interprotein affinity and cofactor activity.
METHODS: The mutations were generated by site-directed mutagenesis. Recombinant FVIII light chain (LC) wild type (WT) and variants were expressed in Bac-to-Bac Baculovirus Expression System and then purified. The activity, stability, and affinity of LC variants were assessed by FXa generation assay and fluorescence anisotropy. The structures of FVIIIa were predicted using PSIPRED (v3.3) and Phyre2.
RESULTS: FXa generation assays revealed that D1903A/E1904A/K1906A/S1907A variants showed 8.4- to 20.3-fold and 3.9- to 5.6-fold increases in apparent dissociation constant for FXase and FVIIIa reconstitution, respectively, compared with WT. Reduced fluorescence anisotropy was observed when fluorescein-Phe-Phe-Arg-FIXa bound to FVIIIa reconstituted with D1903A, E1904A, K1906A, and S1907A variants compared with WT. In both the presence and absence of FIXa, D1903A and E1904A exhibited faster decay rates than WT. While WT FVIIIa showed a 3.26-fold slower decay rate and 0.64-fold shorter half-life in the absence of FIXa compared with its presence, the decay rates and half-lives of FVIIIa reconstituted with D1903A and E1904A remained similar regardless of FIXa presence, suggesting that these mutations abrogate the normal FIXa-mediated stabilization of FVIIIa.
CONCLUSION: Our study identifies FVIIIa residues 1900 to 1908 as key determinants of FIXa binding, advancing understanding of FXase assembly and A3-domain-mediated regulation of complex stability and activity.
OBJECTIVES: This study investigated how individual residues within region 1900 to 1908 contribute to interprotein affinity and cofactor activity.
METHODS: The mutations were generated by site-directed mutagenesis. Recombinant FVIII light chain (LC) wild type (WT) and variants were expressed in Bac-to-Bac Baculovirus Expression System and then purified. The activity, stability, and affinity of LC variants were assessed by FXa generation assay and fluorescence anisotropy. The structures of FVIIIa were predicted using PSIPRED (v3.3) and Phyre2.
RESULTS: FXa generation assays revealed that D1903A/E1904A/K1906A/S1907A variants showed 8.4- to 20.3-fold and 3.9- to 5.6-fold increases in apparent dissociation constant for FXase and FVIIIa reconstitution, respectively, compared with WT. Reduced fluorescence anisotropy was observed when fluorescein-Phe-Phe-Arg-FIXa bound to FVIIIa reconstituted with D1903A, E1904A, K1906A, and S1907A variants compared with WT. In both the presence and absence of FIXa, D1903A and E1904A exhibited faster decay rates than WT. While WT FVIIIa showed a 3.26-fold slower decay rate and 0.64-fold shorter half-life in the absence of FIXa compared with its presence, the decay rates and half-lives of FVIIIa reconstituted with D1903A and E1904A remained similar regardless of FIXa presence, suggesting that these mutations abrogate the normal FIXa-mediated stabilization of FVIIIa.
CONCLUSION: Our study identifies FVIIIa residues 1900 to 1908 as key determinants of FIXa binding, advancing understanding of FXase assembly and A3-domain-mediated regulation of complex stability and activity.