Abstract
Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new beta 15-42 N-terminus. To further understand the interactions between ECs and fibrin beta 15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, beta 1-21; J88B, gamma 63-78) and anti-fibrin (T2G1, beta 15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks beta 1-53. ECs bound the peptide beta 15-42-bovine serum albumin (BSA) conjugate but neither a scrambled beta 15-42 peptide conjugate nor conjugates of beta 24-42, beta 18-27, or beta 18-31. Binding of fibrin-CNBr was inhibited 54% by the beta 15-42-BSA conjugate and 17% by the B beta 1-42-BSA conjugate but not by free beta 15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited > 95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of beta 15-42-BSA by > 75% but not the dose-dependent binding of fibronection to ECs. These data suggest that in their native conformation, FBG B beta 15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of beta 15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.
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