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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1544-1551.)
© 1996 American Heart Association, Inc.

Articles

Heparin-Binding Domain of Fibrin Mediates Its Binding to Endothelial Cells

Tatjana M. Odrljin; Charles W. Francis; Lee Ann Sporn; Leslie A. Bunce; Victor J. Marder; Patricia J. Simpson-Haidaris

the Departments of Medicine (Hematology Unit: T.M.O., C.W.F., L.A.S., L.A.B., V.J.M., P.J.S.-H.), Pathology and Laboratory Medicine (L.A.S., V.J.M., P.J.S.-H.), and Microbiology and Immunology (P.J.S.-H.), University of Rochester School of Medicine and Dentistry, Rochester, NY.

Correspondence to Patricia J. Simpson-Haidaris, PhD, Hematology Unit–Department of Medicine, PO Box 610, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Rochester, NY 14642. E-mail phaida@gigli.medicine.rochester.edu.

Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new ß15-42 N-terminus. To further understand the interactions between ECs and fibrin ß15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, ß1-21; J88B, 63-78) and anti-fibrin (T2G1, ß15-21) 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 ß1-53. ECs bound the peptide ß15-42–bovine serum albumin (BSA) conjugate but neither a scrambled ß15-42 peptide conjugate nor conjugates of ß24-42, ß18-27, or ß18-31. Binding of fibrin-CNBr was inhibited 54% by the ß15-42–BSA conjugate and 17% by the Bß1-42-BSA conjugate but not by free ß15-42 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 ß15-42–BSA by >75% but not the dose-dependent binding of fibronectin to ECs. These data suggest that in their native conformation, FBG Bß15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of ß15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.

Key Words: fibrinogen • proteoglycans • RGDS peptide • cell adhesion • thrombin

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