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 (126I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 °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 × 104 molecules were bound/cell at saturation (∼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 Aα 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.