LOCALIZATION OF A METAL-DEPENDENT EPITOPE TO THE AMINO-TERMINAL RESIDUES-33-40 OF HUMAN FACTOR-IX

Metal binding sites within the Gla domain of vitamin K-dependent coagu lation factors have been divided into nonspecific metal sites and calc ium-specific sites. We demonstrate here that five residues within the Gla domain of factor IX are responsible for the reactivity with the me tal-dependent factor IX monoclonal antibody, A-7. First we demonstrate that modifying any one of three residues within this site in factor I X abolishes the binding of A-7. To confirm the specificity of the anti body, the Gla domain of factor VII was changed at residues 32, 33, 34, 38 and 39 to the homologous residues of human factor IX. These change s were sufficient to generate a factor VII Gla domain with an A-7 bind ing site of the same affinity as that in factor IX. The site identifie d is one of the two major surfaces of the Gla domain and may represent the metal-dependent binding site.