Thrombosis Research, Vol. 80, No. 5, pp. 419-427, 1995 Copyright 0 1995 Elsevier Science&td Printed in the USA. All rights rese&d OD49-3848/95 $9.50 + :km Pergamon LOCALIZATION OF A METAL-DEPENDENT EPITOPE TO THE AMINO TERMINAL RESIDUES 33-40 OF HUMAN FACTOR IX Wing-Fai Cheung#, Alisa S. Wolberg, Darrel W. Stafford, and Kenneth J. Smith* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599 and *Departments of Pathology and Medicine, University of New Mexico, Albuquerque, New Mexico 87131, USA. (Received 22 May 7995 by Editor 5. Dahlb&k; revise&accepted 21 September 1995) Abstract Metal binding sites within the Gla domain of vitamin K-dependent coagulation factors have been divided into nonspecific metal sites and calcium-specific sites. We demonstrate here that five residues within the Gla domain of factor IX are responsible for the reactivity with the metal-dependent factor IX monoclonal antibody, A-7. First we demonstrate that modifying any one of three residues within this site in factor IX abolishes the binding of A-7. To confirm the specificity of the antibody, 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 changes were sufficient to generate a factor VII Gla domain with an A-7 binding site of the same affinity as that in factor IX. The site identified is one of the two major surfaces of the Gla domain and may represent the metal-dependent binding site. The amino-terminal glutamic acid residues of the vitamin K-dependent plasma proteins are modified to y-carboxyglutamic acid (Gla) (1, 2). The Gla domain, containing these modified amino acids, is highly homologous among the vitamin K-dependent proteins. Although the Gla domains can bind various divalent metal ions including calcium, magnesium, strontium, and manganese, only calcium and strontium support reaction with a phospholipid surface and physiological activity (3). The crystal structure of bovine prothrombin fragment 1 (4) shows seven bound calcium ions forming a complex network at the N-terminus of the Gla domain. Furthermore, it was found that eight strontium ions Key words: Factor IX, metal-dependent antibody, epitope mapping #Corresponding author: Wing-fai Cheung, Ph.D., Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Box 575, 5751 23 Uppsala, Sweden. Fax 46-18-550-762. 419