Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372.
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).
Nogami
Keiji
K
Department of Paediatrics, Nara Medical University, Kashihara, Nara, Japan. roc-noga@naramed-u.ac.jp
Saenko
Evgueni L
EL
Takeyama
Masahiro
M
Giddings
John C
JC
Yoshioka
Akira
A
Shima
Midori
M
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*
D000911:Antibodies, Monoclonal; D007518:Isoantibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin