title: Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372.
abstract: FVIII is activated by cleavage at Arg(372), Arg(740), and Arg(1689) by thrombin. This study showed that an anti-A2 monoclonal antibody, with a specific epitope for residues 484-509, and anti-FVIII inhibitor alloantibodies with similar A2 epitopes, inhibited thrombin-catalyzed FVIII activation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that cleavage at Arg(372) but not at Arg(740) occurred at approximately fourfold decreased rate in the presence of anti-A2 antibody. Peptide 484-509 also inhibited co-factor activation, consistent with inhibition of cleavage at Arg(372). Direct binding studies using active-site modified thrombin showed that a 484-509 peptide as well as the anti-A2 antibodies blocked the A2-thrombin binding. Furthermore, covalent cross-linking was observed between the 484-509 peptide and thrombin following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Mutant A2 molecules in which the clustered basic residues in this sequence were converted to alanine were used to assess the binding reactions in a surface plasmon resonance-based assay. Mutants R484A, R489A, R490A, H497A and K499A possessed two to fivefold lower affinity than wild-type A2. These findings demonstrate that clustered basic residues within the 484-509 region of the A2 domain play a part of key role in thrombin-binding, which is responsible for thrombin-catalyzed FVIII activation by cleavage at Arg(372).
authors: Department of Paediatrics, Nara Medical University, Kashihara, Nara, Japan. roc-noga@naramed-u.ac.jp
authors: Keiji
authors: K
authors: Nogami
authors: Evgueni L
authors: EL
authors: Saenko
authors: Masahiro
authors: M
authors: Takeyama
authors: John C
authors: JC
authors: Giddings
authors: Akira
authors: A
authors: Yoshioka
authors: Midori
authors: M
authors: Shima
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007518:Isoantibodies; D010446:Peptide Fragments; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007518:Isoantibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin