Structural and functional analyses of factor VIII
Factor VIII functions as a cofactor for the serine protease factor IXa in the surface-dependent conversion of zymogen factor X to the serine protease, factor Xa. Deficiency of factor VIII activity results in a marked reduction of factor IXa activity and in the subsequent rates of factor Xa generated during the propagation phase of coagulation.
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.
Wakabayashi, H., Zhou, Q., Varfaj, F., and Fay, P. J.
A3 Domain Residue Glu1829 Contributes to A2 Subunit Retention in Factor VIIIa J. Thromb. Haemost., in press.
Varfaj, F., Neuberg, J., Jenkins, P. V., Wakabayashi, H., and Fay, P. J.
Role of P1 Arg in Activated Protein C - Catalyzed Inactivation of Factor VIIIa. Biochemical J. 396, 355-362, 2006.
Wakabayashi, H., Zhou, Q., Nogami#, K., Ansong, C., Varfaj, F., Miles, S., and Fay, P. J.
pH-dependent association of factor VIII chains: Enhancement of affinity at physiological pH by Cu2+. Biochim. Biophys. Acta 1764,1094-1101, 2006.
Nogami, K., Zhou, Q., Myles, T., Leung, L. L. , Wakabayashi, H., and Fay, P. J.
Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain.
J. Biol. Chem. 280, 18476-18487, 2005.
Nogami, K., Zhou, Q., Wakabayashi, H., and Fay, P. J. Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site.
Blood 105, 4362-4368, 2005.
Wakabayashi, H., Su, Y. C. , Ahmad, S. S. , Walsh, P. N. , and Fay, P. J.
A Glu113Ala Mutation within a Factor VIII Ca(2+)-Binding Site Enhances Cofactor Interactions in Factor Xase.
Biochemistry 44, 10298-10304, 2005.
Nogami, K., Wakabayashi, H., Ansong, C., and Fay, P. J. Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit.
Biochim. Biophys. Acta 1701, 25-35, 2004.
Nogami, K., Freas, J., Manithody, C., Wakabayashi, H., Rezaie, A. R. , and Fay, P. J.
Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.
J. Biol. Chem. 279, 33104-33113, 2004.
Nogami K., Lapan K.A., Zhou Q., Wakabayashi H., and Fay P.J.
Identification of a Factor Xa-interactive Site within Residues 337-372 of the Factor VIII Heavy Chain.
J. Biol. Chem. 279, 15763-15771, 2004.
Wakabayashi H., Freas J., Zhou Q., and Fay P.J.
Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity.
J. Biol. Chem. 279, 12677-12684, 2004.
Wakabayashi H., Zhen Z., Schmidt K.M., and Fay P.J.
Mn2+ binding to factor VIII Subunits and its effect on cofactor activity.
Biochemistry 42, 145-153, 2003.
Wakabayashi H., Schmidt K.M., and Fay P.J.
Ca2+ binding to both the heavy and light chains of factor VIII is required for cofactor activity.
Biochemistry 41, 8485-8492, 2002.
Wakabayashi H., Koszelak M., Mastri, M., and Fay P.J Metal-ion independent factor VIII subunit association and the role of calcium and copper for its affinity and activity.
Biochemistry 40, 10293-10300, 2001.