title: Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen.
abstract: OBJECTIVE
  Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen.
  
  
  METHODS
  Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates.
  
  
  RESULTS
  Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]i, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion.
  
  
  CONCLUSIONS
  Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.
authors.affiliation: Department of Clinical Pharmacology, Imperial College School of Medicine, St Mary's Hospital, London, UK.
authors.forename: L
authors.initials: L
authors.lastname: Xie
authors.forename: G F
authors.initials: GF
authors.lastname: Clunn
authors.forename: J S
authors.initials: JS
authors.lastname: Lymn
authors.forename: A D
authors.initials: AD
authors.lastname: Hughes
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology* / Q000502:physiology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009532:Nickel / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D012044:Regression Analysis; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002121:Calcium Channel Blockers; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D016023:Integrins; D009842:Oligopeptides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D014443:Tyrosine; D004533:Egtazic Acid; C047981:arginyl-glycyl-aspartic acid; D009532:Nickel; D005340:Fibrinogen; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium