Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain

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 Arg336 and Arg372 but not at Arg740. Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (Kd: 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479–504 blocked A2 binding by ∼ 80% and ∼ 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 ∼ 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 ∼ 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was ∼ 3-fold lower than that with wild-type A2. These findings demonstrate that Arg484 plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.