title: Proteolysis at Arg740 facilitates subsequent bond cleavages during thrombin-catalyzed activation of factor VIII.
abstract: Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were approximately 50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an approximately 40-fold reduction in the rate of A2 subunit generation, which reflected an approximately 20-fold reduction in cleavage rate at Arg372. Similarly, a approximately 40-fold rate reduction in cleavage at Arg1689 and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and approximately 140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg740 affects cleavage at both Arg372 and Arg1689 sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689, which result in procofactor activation.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 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: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII; D001120:Arginine; D013917:Thrombin; D015951:Factor Xa