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.
