title: Sequences flanking Arg336 in factor VIIIa modulate factor Xa-catalyzed cleavage rates at this site and cofactor function.
abstract: Factor (F)VIII can be activated to FVIIIa by FXa following cleavages at Arg(372), Arg(740), and Arg(1689). FXa also cleaves FVIII/FVIIIa at Arg(336) and Arg(562) resulting in inactivation of the cofactor. These inactivating cleavages occur on a slower time scale than the activating ones. We assessed the contributions to cleavage rate and cofactor function of residues flanking Arg(336), the primary site yielding FVIII(a) inactivation, following replacement of these residues with those flanking the faster-reacting Arg(740) and Arg(372) sites and the slower-reacting Arg(562) site. Replacing P4-P3' residues flanking Arg(336) with those from Arg(372) or Arg(740) resulted in ∼4-6-fold increases in rates of FXa-catalyzed inactivation of FVIIIa, which paralleled the rates of proteolysis at Arg(336). Examination of partial sequence replacements showed a predominant contribution of prime residues flanking the scissile bonds to the enhanced rates. Conversely, replacement of this sequence with residues flanking the slow-reacting Arg(562) site yielded inactivation and cleavage rates that were ∼40% that of the WT values. The capacity for FXa to activate FVIII variants where cleavage at Arg(336) was accelerated due to flanking sequence replacement showed marked reductions in peak activity, whereas reducing the cleavage rate at this site enhanced peak activity. Furthermore, plasma-based thrombin generation assays employing the variants revealed significant reductions in multiple parameter values with acceleration of Arg(336) cleavage suggesting increased down-regulation of FXase. Overall, these results are consistent with a model of competition for activating and inactivating cleavages catalyzed by FXa that is modulated in large part by sequences flanking the scissile bonds.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: DeAngelis
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005169:Factor VIII; D001120:Arginine; D013917:Thrombin; D015951:Factor Xa