Factor VIII (FVIII) is inactivated by limited proteolytic cleavage by plasmin immediately after the activation. However, the plasmin-interactive region(s) in FVIII remain to be determined. Recently, we reported that the A2 domain may interact with plasmin during FVIII inactivation by this protease (Abst #1991,

BLOOD 102, 2002
). In the current study, several approaches were employed to examine the localization and role of plasmin-interactive region(s). Activation and inactivation rate constants of plasmin-catalyzed FVIII and FVIIIa by the addition of isolated A2 subunit were reduced by ~4 and ~13-folds, respectively, in dose-dependent manners using one-stage clotting assay. The addition of Glu-Gly-Arg active-site modified factor IXa, interacts with the A2 domain, also reduced the rate constant of FVIIIa inactivation by ~4-fold. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody 413, recognizing residues 484–509 in factor IXa-interactive site, blocked the plasmin-catalyzed cleavages at Arg336, Arg372, and Arg740 in the heavy chain. Surface plasmon resonance-based assay using anhydro-plasmin, catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that FVIII and isolated A2 subunit bound to anhydro-plasmin with Kd values of 4 and 21 nM, respectively. The binding assay using ELISA with immobilized anhydro-plasmin also showed the similar binding affinities. Monoclonal antibody 413 blocked the A2 subunit binding to anhydro-plasmin by ~80% (IC50: 151 nM). Furthermore, synthetic peptide with sequences 479–504 inhibited this binding by ~55% (Ki: 3 microM), however, peptide with sequences 489–514 had a very weak inhibition (by <20%). To investigate the responsible residues in A2 domain for plasmin binding, the mutant forms of the A2 domain were expressed in baculovirus system and purified. Compared with wild type (23 nM), the affinity of R484A mutant was dramatically decreased by ~250-fold, and the affinities of K377A, K466A, R471A, and K523A mutants also were decreased by 10~40-folds, respectively. Especially, the addition of R484A mutant was reduced inactivation rate constant of plasmin-catalyzed FVIIIa by only ~40% of that of wild type. These findings demonstrate that Arg484 in the A2 domain contains plasmin-binding site responsible for plasmin-catalyzed FVIII(a) inactivation.

Topics:
factor viii, heart sound a2, plasmin, mechlorethamine, monoclonal antibodies, peptides, endopeptidases, factor ixa, peptide hydrolases, serine

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2005, The American Society of Hematology
2005
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