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 calcium-dependent monoclonal antibodies to human factor IX. We demonstrate that the calcium-specific epitope includes residues 1-11 of factor IX, with apparent contributions from other regions of the protein. Antibodies JK.IX -1, -3, and -4 had critical portions of their epitopes in the first eleven amino acids of the Gla domain. These three antibodies could 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 factor 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 terminal tyrosine residue. Furthermore, the calcium-dependent monoclonal antibodies, JK.IX-1, -3, and -4, whose epitope include residues 3 through 11, inhibit factor IX's binding to endothelial cells, for which the binding site on factor IX has been localized to this region. Our results, together with previous studies, confirm the existence of discrete calcium and metal-dependent epitopes within the Gla domain of factor IX and show that the calcium-specific epitope lies near the amino terminus region of this domain.