Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increase s the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A 2 subunit to stimulate factor IXa activity, we show that a panel of these i nhibitors block this activity, inhibition of activity parallels the antibod y potency as measured in the Bethesda assay. These antibodies also block th e A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe- Arg factor IXa. Similar to the IgG fractions, a peptide representing the se quence of the inhibitor epitope (A2 residues 484-509) blocked the A2-depend ent stimulation of factor IXa. These results indicate that antibodies posse ssing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor ac tivity. Furthermore, these results also suggest that factor VIII residues 4 84-509 contribute to a factor IXa-interactive site.