Factor IXa : factor VIIIA interaction - Helix 330-338 of factor IX interacts with residues 558-565 and spatially adjacent regions of the A2 subunit of factor VIIIa

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 implica ted in binding to factor VIIIa. In another study, residues 558-565 of the A 2 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 wit h the 558-565 region of the A2 subunit, Thus, the ability of the isolated A 2 subunit was severely impaired in potentiating factor X activation by IXa( R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330- 338 is replaced by that of factor VII) but it was normal for an epidermal g rowth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth fa ctor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) w ith 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 s ignal upon its binding to the A2 subunit. Apparent K-d(A2) values are: dEGR -IXa(WT) or dEGR-IXa(PCEGF1) similar to 100 nM, dEGR-IXa(R333Q) similar to1 .8 muM, and dEGR-IXa(helixVII) >10 muM. In additional experiments, we measu red the affinities of these factor IXa molecules for a peptide comprising r esidues 558-565 of the A2 subunit, Apparent K-d(peptide) values are: dEGR-I Xa(WT) or dEGR-IXa(PCEGF1) similar to4 muM, and dEGR-IXa(R333Q) similar to 62 muM. 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 similar ly reduced. These data support a conclusion that the helix 330 of factor IX a interacts with the A2 558-565 sequence. This information was used to mode l the interface between the IXa protease domain and the A2 subunit, which i s also provided herein.