Contributions of Active Site Docking and Exosite Binding to the Mechanism of Thrombin-Catalyzed Activation of Factor VIII

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009.Factor VIII is a blood coagulation protein that is defective or deficient in hemophilia A, the most common of severe hereditary bleeding disorders. The activated form of factor VIII, factor VIIIa, serves as a cofactor for the serine protease factor IXa, increasing its catalytic efficiency by several orders of magnitude. Factor VIII is an inactive heterodimeric protein comprised of a heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains). Activation of factor VIII by thrombin occurs through limited proteolysis at residues Arg740 (A2-B domain junction), Arg372 (A1-A2 domain junction), and Arg1689 (a3-A3 junction). Work described here examines how active site and exosite (sites removed from the active site) interactions contribute to the mechanism of thrombin activation of factor VIII. Although there is no known role for cleavage at Arg740, it has been established that the catalytic efficiency of thrombin for cleavage at Arg740 is greater than at either Arg1689 or Arg372. Therefore, the roles for Arg740 and Arg1689 were investigated, along with their effects on the rate limiting cleavage at Arg372, in the mechanism of thrombin-catalyzed cleavage of factor VIII. Four recombinant Bdomainless factor VIII mutants, Arg740His, Arg740Gln, Arg1689His, and Arg1689Gln were prepared and stably expressed in BHK cells. Examination of the Arg740 mutants showed up to an ~140-fold and > 700-fold reduction in the rate for A2 and A3-C1-C2 subunit generation compared to wild-type protein. These data suggest that cleavage at Arg740 affects procofactor activation through facilitating cleavage at both Arg1689 and Arg372. Examination of the Arg1689 variants showed up to a 15-fold reduction in the rate of heavy chain cleavage. This data suggest a linkage between light chain cleavage and cleavages at Arg740 and Arg372 in heavy chain. Overall, these results support a model where cleavage of factor VIII by thrombin is a preferred pathway with cleavage at Arg740 followed by Arg1689, which both influence the activating proteolysis at Arg372. Exosite binding is a major contributor to thrombin proteolysis of factor VIII. The factor VIII a2 segment was investigated as a potential exosite binding site by mutating the acidic residues to Ala or sulfated tyrosines to Phe contained within the region 717-725. Data revealed the sulfated tyrosine residues had little effect on thrombin activation of factor VIII, while the acidic mutants Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala showed decreased rates of thrombin cleavage at each of the three P1 sites. Kinetic data suggested binding affinity (Km) was dominant in reducing catalytic efficiency of the variants. Thus, the data support an exositeinteractive region comprised of residues Glu720, Asp721, Glu724, and Asp725 in the a2 region which contributes to factor VIII activation