title: P3-P3' residues flanking scissile bonds in factor VIII modulate rates of substrate cleavage and procofactor activation by thrombin.
abstract: Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg(372) and Arg(740) in the FVIII heavy chain and Arg(1689) in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg(372), which appears to be rate-limiting. Examination of the P3-P3' residues flanking each P1 site revealed that those sequences at Arg(740) and Arg(1689) are more optimal for thrombin cleavage than at Arg(372), suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg(372) and Arg(1689), respectively. Rates of thrombin cleavage at Arg(372) were increased ~10- and ~3-fold compared with that of wild-type FVIII when it was replaced with P3-P3' residues flanking Arg(740) and Arg(1689), respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg(372) abrogated the need for initial cleavage at Arg(740) to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Newell-Caito
authors.forename: Amy E
authors.initials: AE
authors.lastname: Griffiths
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D002384:Catalysis; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D013379:Substrate Specificity; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
chemical_list: D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin