Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells.
The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C. Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet. Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total. Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM). Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide. In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not. Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain. The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells. Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.
Martinez
J
J
Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Rich
E
E
Barsigian
C
C
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011247:Pregnancy; D011485:Protein Binding; D011503:Transglutaminases / Q000378:metabolism*; D014471:Umbilical Veins
D003432:Cross-Linking Reagents; D009842:Oligopeptides; D010446:Peptide Fragments; D005340:Fibrinogen; D011503:Transglutaminases; D002122:Calcium Chloride