Exosite Interactions Contribute to the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.

Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates cofactors Va and VIIIa. Cleavage of human factor VIIIa occurs at Arg336 and Arg562 located within the A1 and A2 subunits, respectively. While cleavages are not ordered, the former site appears to represent a preferred cleavage site. Efficient catalysis requires binding of APC to a phospholipid surface and to the A3-C1-C2 subunit of factor VIIIa. The latter observation suggests that APC likely binds substrate via an exosite(s) thereby contributing to substrate specificity. A study was undertaken to evaluate contributions of substrate docking at the active site and exosite tethering to the APC catalytic mechanism. Recombinant, human factor VIII mutants where P1 Arg residues at 336 and 562 were substituted with Ala or Gln were constructed and stably expressed. Purified factor VIII was converted to factor VIIIa by thrombin and used as substrate to elucidate the mechanism of cleavages. Proteins mutated at Arg336 were also mutated at Lys338 because the latter residue may serve as an alternative APC cleavage site when residue 336 is mutated. Rates of inactivation of wild type and mutant factor VIIIa molecules and rates of cleavage at Arg336 and Arg562 by APC were monitored in the presence and absence of protein S. The R336A/K338A mutant was inactivated and cleaved at the 336 site approximately 20-fold slower than the wild type, whereas the R336Q/K338Q mutant was completely resistant to cleavage at the 336 site. These results indicate that residues other than Arg may be tolerated at the P1 site, whereas Gln yields a cleavage-resistant substrate. Indeed, the R336Q/K338Q/R562Q (triple Q) mutant was resistant to cleavage at both P1 sites. Furthermore, mutations retarding cleavage at residue 336 showed a dramatic decrease in rates of inactivation suggesting that cleavage at this site correlated with the inactivation of factor VIIIa. The importance of exosite interactions was explored by inhibition experiments examining the inactivation of wild type factor VIIIa in the presence of triple Q mutant factor VIIIa. Wild type factor VIIIa inactivation rates decreased as the proportion of triple Q factor VIIIa increased, indicating that the P1 mutant factor VIIIa effectively sequestered APC from the native substrate. Evaluation of inactivation rates suggested that APC possessed an ~8-fold greater affinity for the triple Q FVIIIa than the wild type factor VIIIa. Consistent with that observation, the Ki for triple Q factor VIIIa (29.5 ± 3.6 nM) was ~5-fold less than the Km for wild type factor VIIIa (133 ± 27 nM). Taken together, these results indicate that mutations in the P1 site that prevent cleavage may also retard dissociation of the enzyme-substrate complex. Overall, results from this study suggest that exosite interactions make a primary contribution to substrate affinity during APC-catalyzed inactivation of factor VIIIa.