Residues 110–126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity

Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.74 microm) within the A1 subunit. Mutagenesis of an acidic residue-rich region in the A1 domain (residues 110-126) homologous to a putative Ca2+ binding site in factor V (Zeibdawi, A. R., and Pryzdial, E. L. (2001) J. Biol. Chem. 276, 19929-19936) and expression of B-domainless factor VIII molecules yielded reagents to probe Ca2+ and Mn2+ binding in a functional assay. Basal activity observed for wild type factor VIII in a metal ion-free buffer was enhanced approximately 2-fold with saturating Ca2+ or Mn2+ and yielded functional K(d) values of 1.2 and 1.40 microm, respectively. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D, D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). These results are consistent with region 110-126 serving a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity.