title: The binding of human factor IX to endothelial cells is mediated by residues 3-11.
abstract: We have used chimeras and point mutations of recombinant coagulation factor IX to examine factor IX's specific interaction with bovine endothelial cells. Previously (Toomey, J. R., Smith, K. J., Roberts, H. R., and Stafford, D. W. (1992) Biochemistry 31, 1806-1808), we restricted the region of factor IX responsible for binding to endothelial cells to its Gla domain. Molecular modeling of the Gla domain of factor IX using the coordinates of the Gla domain of bovine prothrombin-(1-145) (Soriano-Garcia, M., Padmanabhan, K., deVos, A. M., and Tulinsky, A. (1992) Biochemistry 31, 2554-2566) reveals two major surface determinants whose sequences differ among factors IX, X, and VII. A chimeric protein comprised of the Gla domain of factor VII with the remainder of the molecule of factor IX did not bind to the endothelial cell binding site. We changed residues 33, 34, 35, 39, and 40 to those of factor IX without restoring endothelial cell binding. Replacement of amino acid residues 3-10 with those of factor IX restored normal binding. With the knowledge that specific binding was localized to the first 11 amino acids, point mutations were made at residues predicted to be on the surface in this region of the factor IX molecule. Changing lysine 5 to alanine (K5A) or valine 10 to lysine (V10K) resulted in loss of binding with total retention of in vitro clotting activity. The lysine 5 to arginine (K5R) mutation also was fully active in vitro but displayed 3-fold tighter binding. In addition to defining the sequence of factor IX necessary for binding to endothelial cells, these results suggest that the binding site is not phospholipid but instead is specific, and in all likelihood, protein.
authors.affiliation: Department of Biology, University of North Carolina, Chapel Hill 27599.
authors.forename: W F
authors.initials: WF
authors.lastname: Cheung
authors.forename: N
authors.initials: N
authors.lastname: Hamaguchi
authors.forename: K J
authors.initials: KJ
authors.lastname: Smith
authors.forename: D W
authors.initials: DW
authors.lastname: Stafford
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005164:Factor IX / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005164:Factor IX