title: Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain.
abstract: Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484-509), blocked the plasmin-catalyzed cleavage at Arg(336) and Arg(372) but not at Arg(740). Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (K(d): 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479-504 blocked A2 binding by approximately 80% and approximately 55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed approximately 250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10-20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was approximately 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was approximately 3-fold lower than that with wild-type A2. These findings demonstrate that Arg(484) plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.
authors.affiliation: Department of Pediatrics, Nara Medical University, Kashihara, Nara 634-8522, Japan. roc-noga@naramed-u.ac.jp
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nishiya
authors.forename: Evgueni L
authors.initials: EL
authors.lastname: Saenko
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeyama
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanaka
authors.forename: Akira
authors.initials: A
authors.lastname: Yoshioka
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
mesh_terms: D015119:Aminocaproic Acid / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D050505:Mutant Proteins; D010455:Peptides; D021122:Protein Subunits; D005169:Factor VIII; D015949:Factor IXa; D005341:Fibrinolysin; D015119:Aminocaproic Acid