Abstract
Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.
Keywords
factor VIII - thrombin - A3 domain - recombinant - proteolysis
