LOCALIZATION OF A CALCIUM-DEPENDENT EPITOPE TO THE AMINO-TERMINAL REGION OF THE GLA DOMAIN OF HUMAN FACTOR-IX

We have used site-directed mutagenesis to define the epitope of calciu m-dependent monoclonal antibodies to human factor IX. We demonstrate t hat the calcium-specific epitope includes residues 1-11 of factor IX, with apparent contributions from other regions of the protein. Antibod ies JK.IX -1, -3, and -4 had critical portions of their epitopes in th e first eleven amino acids of the Gla domain. These three antibodies c ould bind to a chimera containing the factor VII Gla domain, in which surface residues 3, 4, 5, 9, 10, and 11 were modified to those of fact or IX. In contrast, the epitope of JK.IX-2 was unaffected by mutations in residues 3-11 of factor IX, but was dependent on the amino termina l tyrosine residue. Furthermore, the calcium-dependent monoclonal anti bodies, JK.IX-1, -3, and -4, whose epitope include residues 3 through 11, inhibit factor IX's binding to endothelial cells, for which the bi nding site on factor IX has been localized to this region. Our results , together with previous studies, confirm the existence of discrete ca lcium and metal-dependent epitopes within the Gla domain of factor IX and show that the calcium-specific epitope lies near the amino terminu s region of this domain.