Substitution of Arg(527) and Arg(531) in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function

The functional defect caused bp substitution of Arg(527) (--> Trp) and Arg(531) (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A, For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants, In contrast to FVIII-R527W. the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa. apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg(531) is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg(527) in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr(511)-Leu(530) was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val(517)-Arg(527) are located near the FIXa binding site Ser(558)-Gln(565) Therefore we propose that Arg(527) is part of an extended FIXa binding site, comprising residues Ser(558)-Gln(565) and Val(517)-Arg(527).