Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin

Fibronectin (Fn) binds to fibrin in clots by covalent and noncovalent inter actions. The N- and C-termini of Fn each contain one non-covalent fibrin-bi nding site, which are composed of type 1 (Fl) structural repeats. We have p reviously localized the N-terminal site to the fourth and fifth Fl repeats ((4)Fl.(5)Fl). In the current studies, using proteolytic and recombinant pr oteins representing both the N- and C-terminal fibrin-binding regions, we l ocalized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contri bution of each site to the fibrin-binding activity of intact Fn. By fibrin- affinity chromatography, a protein composed of the (10)Fl repeat through to the C-terminus of Fn ((10)Fl-COOH), expressed in COS-1 cells, and (10)Fl-( 12)Fl, produced in Saccharomyces cerevisiae, displayed fibrin-binding activ ity. However, since (10)Fl and (10)Fl.(11)Fl were not active, the presence of (12)Fl is required for fibrin binding. A proteolytic fragment of 14.4 kD a, beginning 14 residues N-terminal to (10)Fl, was isolated from the fibrin -affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein ( FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated w ells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs fr om the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn b y ELISA yielded estimated K-d values of 216, 18 and 2.1 nM, respectively. T he higher fibrin-binding affinity of the N-terminus was substantiated by th e ability of both a recombinant (4)Fl.(5)Fl and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80% , and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by a bsorption with the 25.9 kDa FBP. We propose that whereas the N-terminal sit e appears to contribute to most of the binding activity of native Fn to fib rin, the specific binding of the C-terminal sire may strengthen this intera ction.