IDENTIFICATION OF RESIDUES IN THE GLA-DOMAIN OF HUMAN FACTOR-IX INVOLVED IN THE BINDING TO CONFORMATION SPECIFIC ANTIBODIES

The binding of Ca2+ induces a conformational change in factor IX which can be monitored with conformation specific antibodies. Anti-FIX:Mg(I I) antibodies recognize a conformational epitope (FIX') that can be in duced by several metal ions such as Ca2+, Mg2+, Mn2+ and Ba2+, while a nti-FIX:Ca(II) antibodies recognize a conformational epitope (FIX)tha t can be only induced by Ca2+ and Sr2+ ions (Liebman et al., J. Biol. Chem., vol. 262 (1987) pp. 7605-7612). The latter conformation is esse ntial for the function of factor IX. In this study we tried to identif y residues in the Gla-domain of factor IX which are involved in bindin g to anti-factor IX:Mg(II) and anti-factor IX:Ca(II) antibodies. For t his we substituted residues in recombinant human factor IX for those o f factor X or factor VII. The substitution of residues 1-40 of factor IX by those of factor VII eliminated binding to both types of antibodi es, Re-introduction of factor IX specific residues increased the bindi ng to conformation specific anti-factor IX antibodies, but reduced the binding to conformation specific anti-factor VII antibodies, indicati ng that the structural integrity of the Gla-domain was not seriously a ffected by the mutations. We provide evidence that residues 33, 39 and 40 of human factor IX are important for binding to anti-factor IX:Mg( II) antibodies, while residues 1-11 are important for binding to anti- factor IX:Ca(II) antibodies. (C) 1998 Elsevier Science B.V.