HEPARIN-BINDING DOMAIN OF FIBRIN MEDIATES ITS BINDING TO ENDOTHELIAL-CELLS

Spreading of human umbilical vein endothelial cells (ECs) on fibrin re quires thrombin cleavage of fibrinopeptide B (FPB) and subsequent expo sure of the new beta 15-42 N-terminus. To further understand the inter actions 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, gamm a 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 ligan d. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspensi on in a dose-dependent and saturable manner. By contrast, FBG-CNBr bou nd only 50% as well as EC monolayers, with no significant binding of i ntact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fr agment E, which lacks beta 1-53. ECs bound the peptide beta 15-42-bovi ne-42 serum albumin (BSA) conjugate but neither a scrambled beta 15-42 peptide conjugate nor conjugates of beta 24-42, beta 18-27, or beta 1 8-31. Binding of fibrin-CNBr was inhibited 54% by the beta 15-42-BSA c onjugate 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 wa s inhibited >95% by heparin in a concentration-dependent manner; the s ame concentrations of heparin inhibited binding of beta 15-42-BSA by > 75% but not the dose-dependent binding of fibronectin to ECs. These da ta suggest that in their native conformation, FBG B beta 15-42 sequenc es are unavailable for binding to ECs and that thrombin-induced exposu re of beta 15-42 is required for binding by a heparin-dependent, RGD-i ndependent mechanism at the new N-terminus of fibrin.