Factor IXa:Factor VIIIa Interaction

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca2+ and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558–565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558–565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXaR333Q and by a helix replacement mutant (IXahelixVII in which helix 330–338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXaPCEGF1 in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent Kd(A2) values are: dEGR-IXaWT or dEGR-IXaPCEGF1 ∼100 nm, dEGR-IXaR333Q ∼1.8 μm, and dEGR-IXahelixVII >10 μm. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558–565 of the A2 subunit. ApparentKd(peptide) values are: dEGR-IXaWTor dEGR-IXaPCEGF1 ∼4 μm, and dEGR-IXaR333Q ∼62 μm. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558–565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558–565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.