Heparin-binding domain of fibrin mediates its binding to endothelial cells

Abstract

Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new β15-42 N-terminus. To further understand the interactions between ECs and fibrin β15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, β1-21; J88B, γ63-78) and anti-fibrin (T2G1, β15-21) 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 β1-53. ECs bound the peptide β15-42-bovine serum albumin (BSA) conjugate but neither a scrambled β15-42 peptide conjugate nor conjugates of β24-42, β18-27, or β18-31. Binding of fibrin-CNBr was inhibited 54% by the β15-42-BSA conjugate and 17% by the Bβ1-42-BSA conjugate but not by free β15-42 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 β15-42-BSA by >75% but not the dose-dependent binding of fibronectin to ECs. These data suggest that in their native conformation, FBG Bβ15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of β15-42 is required for binding by a heparin dependent, RGD-independent mechanism at the new N-terminus of fibrin.