title: Acidic residues C-terminal to the A2 domain facilitate thrombin-catalyzed activation of factor VIII.
abstract: Factor VIII is activated by thrombin through proteolysis at Arg740, Arg372, and Arg1689. One region implicated in this exosite-dependent interaction is the factor VIII a2 segment (residues 711-740) separating the A2 and B domains. Residues 717-725 (DYYEDSYED) within this region consist of five acidic residues and three sulfo-Tyr residues, thus representing a high density of negative charge potential. The contributions of these residues to thrombin-catalyzed activation of factor VIII were assessed following mutagenesis of acidic residues to Ala or Tyr residues to Phe and expression and purification of the B-domainless proteins from stable-expressing cell lines. All mutations showed reduced specific activity from approximately 30% to approximately 70% of the wild-type value. While replacement of the Tyr residues showed little, if any, effect on rates of thrombin-catalyzed proteolysis of factor VIII and consequent activation, the acidic to Ala mutations Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala showed decreased rates of proteolysis at each of the three P1 residues. Mutations at residues Glu724 and Asp725 were most affected with double mutations at these sites showing approximately 10-fold and approximately 30-fold reduced rates of cleavage at Arg372 and Arg1689, respectively. Factor VIII activation profiles paralleled the results assessing rates of proteolysis. Kinetic analyses revealed these mutations minimally affected apparent V max for thrombin-catalyzed cleavage but variably increased the K m for procofactor up to 7-fold, suggesting the latter parameter was dominant in reducing catalytic efficiency. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 likely constitute an exosite-interactive region in factor VIII facilitating cleavages for procofactor activation.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Newell
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D002384:Catalysis; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin