My research focuses on the role of metal ions for structural integrity and function of factor VIII. Active factor VIII can be reconstituted by combining the isolated subunits in the presence of Ca2+ or Mn2+. Ca2+ had little effect on inter-subunit affinity yet converted the inactive dimer to an active form. Alternatively, Cu2+ enhanced the inter-subunit affinity ~100-fold but yielded a dimer lacking cofactor activity. Site-directed mutagenesis is employed to identify residues important in Ca2+ binding to factor VIII and resultant effects of this binding on activity. Several acidic amino acids within segment 110-126 of the A1 domain of factor VIII that likely participate in the coordination of Ca2+ necessary for generation of maximal cofactor specific activity.
