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 th ese residues is associated with mild haemophilia A, For both FVIII-R527W an d FVIII-R531H, activity was lower than antigen, indicating a functional def ect for both variants, In contrast to FVIII-R527W. the amount of FVIII-R531 H heterodimer present in plasma was reduced compared to heavy and light cha in 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 stabil ity of both the heterodimer and the activated FVIII heterotrimer. Recombina nt 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-d erived synthetic peptide Tyr(511)-Leu(530) was able to inhibit FX activatio n and that this inhibition could be overcome by addition of increasing conc entrations of FIXa. Furthermore, in the three-dimensional FVIII model resid ues Val(517)-Arg(527) are located near the FIXa binding site Ser(558)-Gln(5 65) 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).