Previous studies revealed that cleavage at Arg-318-Ser-319 in the
protease domain
autolysis loop of
factor IXa results in its diminished binding to
factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330-338 (162-170 in
chymotrypsin numbering) of IXa in its interaction with VIIIa. IXWT, eight point mutants mostly based on
hemophilia B patients, and a replacement mutant (IXhelixVII in which helix 330-338 is replaced by that of
factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-
tissue factor-Ca2+ or XIa-Ca2+. However, in both the presence and absence of
phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IXQ50P and IXPCEGF1, in which EGF1 domain is replaced by that of
protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of
phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the
protease domain above the
phospholipid surface for optimal interaction with VIIIa.