Protease and EGF1 domains of factor IXa play distinct roles in binding to factor VIIIa - Importance of helix 330 (helix 162 in chymotrypsin) of protease domain of factor IXa in its interaction with factor VIIIa

Previous studies revealed that cleavage at Arg-318-Ser-319 in the protease domain autolysis loop of factor Ma results in its diminished binding to fac tor VIIIa. Now, we have investigated the importance of adjacent surface-exp osed helix 330-338 (162-170 in chymotrypsin numbering) of IX in its interac tion with VIIIa, IXWT, eight point mutants mostly based on hemophilia B pat ients, and a replacement mutant (IXhelixVII in which helix 330-338 is repla ced by that of factor VII) were expressed, purified, and characterized. Eac h mutant was activated normally by VIIa-tissue factor-Ca2+ or XIa-Ca2+, How ever, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IX EGF1 domain in bi nding to VIIIa was also examined, Two mutants (IXQ50P and IXPCEGF1, in whic h EGF1 domain is replaced by that of protein C) were used. Strikingly, inte ractions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in Ma provides a crit ical binding site for VIIIa and that the EGF1 domain in this context primar ily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.