Polymerization Site in the β Chain of Fibrin: Mapping of the Bβ1-55 Sequence

Document Type

Article

Publication Title

Biochemistry

Abstract

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the Bβ31–42 and /315-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B/31-42 contained a fibrin polymerization site. In other to identify and map the site, the peptides B/31-42 and /315-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: /315-18, /315-26, /324-42, /340-54, /350-55, and al 7–19-Pro. While B/31-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, /315-42, was a strong inhibitor. The peptides /315-18, /315-26, and /315-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides 024–42, 040–54, and 050–55 were inactive. Computer-aided prediction of probable secondary structures in B/31-55, together with polymerization inhibition by /315-55-derived peptides, suggested that the polymerization site in the amino-terminal domain of the 0 chain might be composed of noncontiguous amino acids. However, the NH2-terminal disulfide knot of fibrin, which contains amino termini of the a, 0, and y chains, not only was a stronger inhibitor of polymerization than /315-42, but it also had a higher affinity for binding to fibrin monomer. Therefore, it is proposed that the fibrin polymerization site in the NH2-terminal domain of fibrin may be composed of sequence derived from both the a and 0 chains. © 1991, American Chemical Society. All rights reserved.

First Page

162

Last Page

168

DOI

10.1021/bi00215a024

Publication Date

11-1-1991

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