Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to gamma-chain residues 337-379.

Abstract

Human fibrinogen and the plasmin-generated fibrinogen fragment D were photoaffinity labeled specifically with the peptide [14C]Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide. In the case of fibrinogen, >85% of the incorporated radioactivity was found in the y chain. Similarly, when fragment D (Mr, 90,000) was labeled with the same derivatized peptide, virtually all the radioactivity was found in the y-chain portion. The labeled fragment D was treated with CNBr and an initial purification was achieved by two gel.filtration steps. The labeled material was purified further by HPLC and was also compared with CNBr digests of unlabeled material. Amino acid analysis and gas-phase sequencing showed the labeled fragment to be y-chain residues 337-379.In vertebrates, the conversion of fibrinogen into fibrin is initiated by the thrombin-catalyzed removal of the fibrinopeptides A, the immediate consequence of which is the exposure of a pair of new amino termini beginning with the sequence Gly-Pro-Arg (for a review, see ref. 1). Synthetic peptides beginning with that sequence bind strongly to fibrinogen and are able to block fibrin formation (2). The binding has been localized to the terminal domains corresponding to the large plasmin-generated fragment designated D1 (3). In contrast, synthetic peptides beginning with the sequence Gly-His-Arg, which corresponds to the amino terminus of chains after release of the fibrinopeptides B, do not block fibrin formation, even though they do bind to fibrinogen. The Gly-His-Arg peptides bind to both fragments D1 and D3, the latter being a further degradation product of the former. Peptides with the sequence Gly-Pro-Arg do not bind to fragment D3, however, which lacks the carboxylterminal 109 residues of the y chain, leading to the supposition that the complementary binding site for the Gly-Pro-Arg "knob" is situated in the carboxyl-terminal third of the y-chain portion of fragment D (4). Previous efforts to localize further the complementary binding sites for the two sets of peptides have been a matter of dispute. Thus, Olexa and Budzynski (5) reported that the carboxylterminal 38 residues ofthe y chain (i.e., 'y374411) embodied the principal binding site; a similar claim about residues y374-396 was made by Horwitz et aL (6). Subsequently, Southan et al. (7) and Varadi and Scheraga (8) reported experiments indicating that these results were likely artifactual. We now report the results of a photoaffinity labeling study with the peptide Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide that implicates residues 337-379 from the y chain. MATERIALS AND METHODSHuman fibrinogen was prepared from blood bank plasma as described (9). Fragment D was prepared by digesting the fibrinogen with plasmin in the presence of calcium ions (10), thereby limiting the digest to the large molecular weight D usually referred to as fragment D1. Digestions were stopped by the addition of the plasmin inhibitor Trasylol (aprotinin from bovine lung), and fragments were purified either by ion-exchange chromato...

Clinical features and molecular basis of 102 Chinese patients with congenital dysfibrinogenemia

“…This may also be the potential mechanism of dysfibrinogenemia caused by novel γAsn334(308)Thr and γGly335(309)Cys. γTrp395(369)Leu was supposed to be associated with defective fibrin polymerization since γTrp395(369) was in pocket a, which is localized to residues 337 to 379 of mature γ chain [29]. Moderate correlations between Fg:C and MA or MA-CFF have been widely identified [8][9][10], as well as in normal individuals in the current study.…”

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of γ-irradiation

“…There are two polymerization sites, designed 'A' and 'B', uncovered after the fibrinopeptides release from the N-termini of a and b chains, respectively. The 'A' sites bind to complementary 'a' binding pockets, localized to residues 337-379 of the C-terminal of the g chain and close to Tyr363 [27]. The 'A: a' interaction results in the end-to-end polymerization of fibrin monomers to protofibrils.…”

Crystal Structure of Fragment Double-D from Human Fibrin with Two Different Bound Ligands,

“…Thus, synthetic Gly-Pro-Arg peptides do not bind to fragment D in which the -chain is intact but in which the γ-chain has been degraded at the carboxyl end (4,5). Also, photoaffinity labeling experiments with Gly-Pro-Arg derivative peptides led to the exclusive labeling of Tyr γ363 (6,7). Finally, X-ray structures of two different kinds of fragment preparations cocrystallized or soaked with synthetic Gly-Pro-Arg-Pro-amide showed the ligand bound to a hole on the γ-chain globular domain.…”