Contribution of P4-P3′ Residues Surrounding P1 Residues Arg336 and Arg562 in the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.

The essential role of factor VIII in blood coagulation is evident from the bleeding diathesis hemophilia A, which results from a deficiency or defect in factor VIII. Activated factor VIII (factor VIIIa) serves as a cofactor for factor IXa in the factor Xase complex, which activates factor X during the propagation phase of coagulation. Factor VIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3–C1–C2 subunits. Down-regulation of factor Xase is achieved by cofactor inactivation and is thought to occur by a non-proteolytic mechanism involving dissociation of the A2 subunit as well as a proteolytic mechanism catalyzed by activated protein C (APC). APC cleaves the P1 residues Arg336 near the C-terminus of the A1 subunit and Arg562 bisecting the A2 subunit. We recently demonstrated that these cleavages occur in an independent non-sequential fashion, with residue Arg336 being cleaved at a rate ~25-fold faster than Arg562 (Varfaj et al., Biochem J. 2006). While substantial evidence implicates involvement of exosite-directed interactions in the catalytic mechanism of APC, another factor that may contribute to the disparate cleavage rates are residues surrounding the P1 Arg residues. To examine the roles of these sequences in cofactor cleavage, we prepared two factor VIII mutants where the P2–P4 and P1′–P3′ residues surrounding Arg336 (Pro-Gln-Leu and Met-Lys-Asn, respectively) were replaced with those residues surrounding Arg562 (Val-Asp-Gln and Gly-Asn-Gln, respectively), and designated Arg336P2-P4A2 and Arg336P1′-P3′A2. In addition, a single mutant was prepared where the P4-P3′ residues surrounding Arg562 were replaced with those residues surrounding Arg336, and designated Arg562P4-P3′A1. Recombinant, B-domainless factor VIII proteins were stably expressed in BHK cells and purified. Specific activity values measured for Arg336P2-P4A2 and Arg336P1′-P3′A2 mutants were similar to that of wild type (WT) factor VIII, whereas Arg562P4-P3′A1 showed a specific activity value <1% that of WT factor VIII. This latter observation was consistent with the substitution of the A1 residues altering the factor IXa-interactive site contained within A2 residues 558–565. Western blot analysis examining the rates of APC-catalyzed cleavage at Arg336 showed an ~60-fold reduction for the Arg336P2-P4A2 mutant and an ~10-fold reduction for Arg336P1′-P3′A2 mutant compared to cleavage rates observed for WT factor VIIIa. Rates of cleavage at the Arg562 site in these mutants were similar to the WT protein at this site. These results suggest that the native sequence surrounding Arg336 possesses residues more optimal for cleavage by APC than those that surround Arg562 in the A2 subunit. Examination of the Arg562P4-P3′A1 mutant showed a modest increase (~2-fold) in cleavage rate at Arg562, whereas cleavage at the Arg336 was similar to the WT control. Overall, these results suggest a relatively minor role for specific sequences in the cleavage mechanism for APC at the A2 site, whereas a more dominant role for sequence specificity appears necessary for efficient proteolysis at the A1 site, which represents the primary site of attack by APC.