The fibrin B?125-135 site is involved in the lateral association of protofibrils

Abstract

Earlier we reported that during the human fibrinogen to fibrin transition a neoantigenic determinant was exposed in the Bβ119-133 fragment, where a hinge locus is situated. The fibrin-specific mAb FnI-3c and its Fab-fragment with epitope in this fragment inhibited the lateral association of protofibrils. We suggested that the epitope coincided with a site involved in this process. In this work we investigated the epitope location more precisely and defined a functional role for its exposure in the hinge locus of the molecule. It was found that mAb FnI-3c bound to human, horse and rabbit fibrins, all of which have Lys in the position corresponding to human BβK130, but not to bovine and rat fibrins, which have other amino acid residues in this position, strongly suggesting that BβK130 provides the integral part of the epitope. This fact, homology data, and structural biological analysis of the amino acid sequences around BβK130 indicate that the site of interest is localized within Bβ125-135. The synthetic peptides Bβ121-138 and Bβ125-135, unlike their scrambled versions, bound to mAb FnI-3c in SPR analysis. Both peptides, but not their scrambled versions, inhibited the lateral association of protofibrils. The FnI-3c epitope is exposed after fibrinopeptide A cleavage and desA fibrin monomer formation. Structural biological analysis of the fibrinogen to fibrin transition showed a distinct increase of flexibility in the hinge locus. We propose that the structural transformation in the fibrin hinge regions leads to the conformation necessary for lateral association of protofibrils. K e y w o r d s: fibrinogen to fibrin transition, coiled-coil connector, protofibril lateral association, hinge region, neoantigenic determinant. introduction Fibrinogen is a dimer, with each subunit of the molecule being formed by three polypeptide chains: Aα, Bβ and γ. The molecule consists of a central E, two peripheral D, and two extended αC regions [1]. The central E-region consisting of (Aα1-104, Bβ1-133, γ1-72) 2 is connected to the two peripheral Dregions (Aα105-219, Bβ134-461, γ73-411) by two long flexible coiled-coil connectors (Aα48-161, Bβ79-193, γ23-135). The N-terminal parts of the connectors (Aα48-104, Bβ79-133, γ23-62) belong to the E region and the C-terminal parts (Aα105-161, Bβ134-193, γ63-135) to the D regions. A hinge locus (α99-110, β130-155, γ70-100) is located in the middle part of the coiled-coil region [2, 3]. The extended αC regions (Aα220-610) consist of unfolded flexible segments (Aα220-391) and more structured αC-domains (Aα392-610). Intermolecular binding of the fibrin polymerization sites A:a as well as putative C:c sites [4], leads to protofibril formation. The protofibrils associate laterally, initially forming fibrils and, subsequently, a three-dimensional fibrin net. At the stage of protofibrils and fibril formation, inter-protofibril binding

Thrombosis-associated hypofibrinogenemia: novel abnormal fibrinogen variant FGG c.8G>A with oxidative posttranslational modifications

“…Mapping of the Bβ chain indicated that all structural parts of this chain can be oxidized [56]. As described previously, superhelical regions of fibrinogen also influence lateral aggregation of protofibrils [57,58]. Modifications of amino acids side chains change their spatial structure, and, thereby inhibit lateral aggregation [59,60].…”

Probing Fibrinogen Structure: Insights From Limited Proteolysis, Peptides, and Monoclonal Antibodies

“…Scheme of fibrinogen molecule with some effectors (antibody, protease, peptide) targeted to the distinct parts of the molecule. Modified after: [13] I-3C antibody targeted to 122-134 residue of B-chain of fibrinogen[14]; BK134-G135 is a peptide bond that is cleaved during the formation of TSK-fragment of fibrinogen[15]; B125-135 is a synthetic peptide [16]; I-5A antibody targeted to 509-610 residue of A-chain of fibrinogen[17]; AA504-S505 -is a peptide bond that is cleaved by protease II from the culture medium of Bacillus thuringiensis[18]; AA414-610 is a peptide generated by protease from the venom of Gloydius halys halys[19]; I-3B antibody targeted to 15-53 residue of B-chain of fibrinogen[20]; BR42-A43 is a peptide bond that is cleaved by protease II from the venom of Echis multisquamatis[21]; B15-118 is a synthetic peptide[22]; II-4d antibody targeted to 86-240 residue of -chain of fibrinogen[23]; 69-77 is a synthetic peptide[7]; III-3B antibody targeted to 155-160 residue of B-chain of fibrinogen[24].…”