Synthetic peptides and fragments cleaved from native fibrinogen are used in studies to localize binding sites for various ligands. We addressed the question how the native conformation of a selected γ chain segment is affected by scission of the original chain. The conformation of the γ chain COOH-terminus of intact fibrinogen and its various fragments containing this region has been compared by an immunochemical analysis. An antibody population specific for the native epitope within the γ391-405 segment was isolated by affinity chromatography on the corresponding synthetic peptide. Between 19.2 and 22.8% of antibodies were obtained from three different antisera indicating that this region represents one of the major epitopes of native fibrinogen. Anti-γ391-405 antibodies were used to determine the value of Kconf the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, γ chain and γ391-405 synthetic peptide. In addition, the effect of 5 M guanidine-HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, and quantitative analytical parameters, CI50% and CIs, calculated from competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.3% of fragment D1, 8.9% of fragment DD, 3.6% of the γ chain and less than 0.008% of the γ391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine-HCl affected only slightly the conformation of this γ chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, crosslinking site, and platelet recognition site.
Electron microscopy of fibrin Paris I