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

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 Arg372. We have also found A1 of FVIII bound to anion‐binding exosite I thrombin. The present study focused, therefore, on thrombin interaction with residues 337‐372 containing clustered acidic and hirugen‐like sequences.AimTo identify specific site(s) within region FVIII.Methods ResultsThe synthetic peptide 337‐353 sulfated Tyr346 (337‐353S) significantly blocked activation Arg372, while a corresponding 354‐372 had no significant effect. Treatment 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide cross‐link 340‐350S suggested 344‐349 was involved in interaction. Alanine‐substituted mutants, Y346A D347A/D348A/D349A, depressed peak ~ 50% rates 10% 20% compared wild type (WT). level rate Arg372 using mutants 337‐346 substituted hirugen‐sequences (MKNNEEAEDY337‐346GDFEEIPEEY) were 1.5‐ 2.5‐fold WT, respectively. Surface plasmon resonance‐based analysis demonstrated Kd active‐site modified interactions D347A/D348A/D349A 3‐ 6‐fold higher than hirugen‐hybrid mutant facilitated association kinetics 1.8‐fold WT.ConclusionResidues 346‐349 Tyr provided site A showed more efficient