The C-terminal acidic region in the A1 domain of factor VIII facilitates thrombin-catalyzed activation and cleavage at Arg372

Background: Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin-interactive sites responsible for cleavage at Arg(372). We have also found that the A1 domain of FVIII bound to the anion-binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337-372 containing clustered acidic and hirugen-like sequences. Aim: To identify specific thrombin-interactive site(s) within the A1 acidic region of FVIII. Methods and Results: The synthetic peptide of residues 337-353 with sulfated Tyr(346) (337-353S) significantly blocked thrombin-catalyzed FVIII activation and cleavage at Arg(372), while a corresponding peptide of residues 354-372 had no significant effect. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin and 340-350S suggested that the 344-349 clustered acidic region was involved in thrombin interaction. Alanine-substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin-catalyzed activation and cleavage at Arg(372), with peak activation at similar to 50% and cleavage rates of similar to 10% to 20% compared to wild type (WT). The peak level of thrombin-catalyzed activation and the cleavage rate at Arg(372) using FVIII mutants with 337-346 residues substituted with hirugen-sequences (MKNNEEAEDY337-346GDFEEIPEEY) were similar to 1.5- and similar to 2.5-fold of WT, respectively. Surface plasmon resonance-based analysis demonstrated that the K-d for active-site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was similar to 3- to 6-fold higher than that of WT, and that the hirugen-hybrid mutant facilitated association kinetics similar to 1.8-fold of WT. Conclusion: Residues 346-349 with sulfated Tyr provided a thrombin-interactive site responsible for activation and cleavage at Arg(372). A hirugen-hybrid A1 mutant showed more efficient thrombin-catalyzed cleavage at Arg(372).