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.
Highlights
FXa initially activates FVIII and subsequently inactivates FVIII/FVIIIa by proteolysis at specific cleavage sites
Specific sequences that flank Arg336 exhibit a significant impact on the generation of thrombin in plasma. These results indicate that cleavage of factor VIII and VIIIa at Arg336 by factor Xa is modulated by sequences flanking that scissile bond, and that replacing the authentic sequence with those sequences flanking other scissile bonds that are native to the protein and associated with procofactor activation, yield significant acceleration in the rates of inactivation
Factor Xa exhibits a dual activity toward factor VIII
Summary
FXa initially activates FVIII and subsequently inactivates FVIII/FVIIIa by proteolysis at specific cleavage sites. Plasma-based thrombin generation assays employing the variants revealed significant reductions in multiple parameter values with acceleration of Arg336 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. Either slowing cleavage at this site by replacement with the Arg562 flanking sequence or eliminating this cleavage by an R336Q mutation yielded increased cofactor activity and enhanced thrombin generation These data indicate that sequences flanking the scissile bonds in factor VIII attacked by factor Xa make a significant contribution to proteolytic rates and influence activating/inactivating pathways catalyzed by this enzyme
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