Abstract
BackgroundFactor 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 Arg372. 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. AimTo identify specific thrombin‐interactive site(s) within the A1 acidic region of FVIII. Methods and ResultsThe synthetic peptide of residues 337‐353 with sulfated Tyr346 (337‐353S) significantly blocked thrombin‐catalyzed FVIII activation and cleavage at Arg372, 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 Arg372, with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin‐catalyzed activation and the cleavage rate at Arg372 using FVIII mutants with 337‐346 residues substituted with hirugen‐sequences (MKNNEEAEDY337‐346GDFEEIPEEY) were ~ 1.5‐ and ~ 2.5‐fold of WT, respectively. Surface plasmon resonance‐based analysis demonstrated that the Kd for active‐site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3‐ to 6‐fold higher than that of WT, and that the hirugen‐hybrid mutant facilitated association kinetics ~ 1.8‐fold of WT. ConclusionResidues 346‐349 with sulfated Tyr provided a thrombin‐interactive site responsible for activation and cleavage at Arg372. A hirugen‐hybrid A1 mutant showed more efficient thrombin‐catalyzed cleavage at Arg372.
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