title: Polymerization site a function dependence on structural integrity of its nearby calcium binding site.
abstract: To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: gamma D364A in the a site and gamma D318A, gamma D320A, and gamma D318 + gamma D320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS-PAGE analysis of the products showed that gamma D364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected gamma D318A, gamma D320A, or gamma D318 + gamma D320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Lounes
authors.forename: N
authors.initials: N
authors.lastname: Okumura
authors.forename: K A
authors.initials: KA
authors.lastname: Hogan
authors.forename: L
authors.initials: L
authors.lastname: Ping
authors.forename: S T
authors.initials: ST
authors.lastname: Lord
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005340:Fibrinogen; D002118:Calcium