Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (

). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.

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