The Mechanisms for Catalytic Inactivation of FVIIIa by APC and Factor Xa

Factor VIII (FVIII) is activated by thrombin and factor Xa (FXa) through proteolysis at Arg372, Arg740 and Arg1689 and inactivated by activated protein C (APC) and FXa through cleavage at Arg336 and Arg562. Studies presented here examine FVIIIa inactivation by APC and FXa through observing contributions of active site (residues flanking cleavage sites) and exosite (residues removed from cleavage sites) interactions to rates of cleavage at Arg336, the dominant inactivating reaction.;Residues flanking FVIII(a) cleavage sites contribute to the rate of cleavage as replacing residues flanking Arg336 with those flanking Arg562 greatly reduced Arg336 cleavage rates by APC (Varfaj et al, J. Biol. Chem. 2007). The role of individual residues flanking Arg336 (residues 333-339) was assessed using FVIII proteins containing point mutations based upon those flanking Arg562 of FVIII or Arg506 of FV. Results demonstrated reductions in Arg336 cleavage and cofactor inactivation rates for most of the mutants indicating residues flanking Arg336 are in general more favorable than those surrounding Arg562 and appear to have a synergistic effect on the faster rate of cleavage at this site.;FXa activates FVIII and subsequently inactivates FVIII(a) at a slower rate. We assessed the contributions of residues flanking Arg336 to cleavage rate and cofactor function following replacement of these residues with those flanking faster-reacting Arg740 and Arg372 and the slower-reacting Arg562 site. Results showed increases in FXa cleavage rates at Arg336 when flanking residues were mutated to Arg372 or Arg740. Furthermore, these variants exhibited reductions in peak activity and thrombin levels following FXa activation and plasma-based thrombin generation assays, respectively. The reverse effect was seen for the 336(P4-P3')562 variant. This data suggests a model for competing activating and inactivating FVIII(a) cleavage reactions catalyzed by FX that are modulated by residues flanking scissile bonds, and furthermore, that FX may be a self-regulating enzyme.;The basic APC exosite interacts with acidic regions on FVIII and determines substrate specificity. FVIII proteins where acidic residues within the C-terminus of the FVIIIa A2 domain (residues 717-725) were mutated to Ala were employed to identify this as an exosite interactive region. Kinetic parameters suggest the APC exosite interacts with this region.