Fibrinogen and fibrin play important, overlapping roles in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, and neoplasia. These events are regulated to a large extent by fibrin formation itself and by complementary interactions between specific binding sites on fibrin(ogen) and extrinsic molecules including proenzymes, clotting factors, enzyme inhibitors, and cell receptors. Fibrinogen is comprised of two sets of three polypeptide chains termed Aα, Bβ, and γ, that are joined by disulfide bridging within the N‐terminal E domain. The molecules are elongated 45‐nm structures consisting of two outer D domains, each connected to a central E domain by a coiled‐coil segment. These domains contain constitutive binding sites that participate in fibrinogen conversion to fibrin, fibrin assembly, crosslinking, and platelet interactions (e.g., thrombin substrate, Da, Db, γXL, D:D, αC, γA chain platelet receptor) as well as sites that are available after fibrinopeptide cleavage (e.g., E domain low affinity non‐substrate thrombin binding site); or that become exposed as a consequence of the polymerization process (e.g., tPA‐dependent plasminogen activation). A constitutive plasma factor XIII binding site and a high affinity non‐substrate thrombin binding site are located on variant γ′ chains that comprise a minor proportion of the γ chain population. Initiation of fibrin assembly by thrombin‐mediated cleavage of fibrinopeptide A from Aα chains exposes two EA polymerization sites, and subsequent fibrinopeptide B cleavage exposes two EB polymerization sites that can also interact with platelets, fibroblasts, and endothelial cells. Fibrin generation leads to end‐to‐middle intermolecular Da to EA associations, resulting in linear double‐stranded fibrils and equilaterally branched trimolecular fibril junctions. Side‐to‐side fibril convergence results in bilateral network branches and multistranded thick fiber cables. Concomitantly, factor XIII or thrombin‐activated factor XIIIa introduce intermolecular covalent ε‐(γ glutamyl)lysine bonds into these polymers, first creating γ dimers between properly aligned C‐terminal γXL sites, which are positioned transversely between the two strands of each fibrin fibril. Later, crosslinks form mainly between complementary sites on γ chains (forming γ‐polymers), and even more slowly among γ dimers to create higher order crosslinked γ trimers and tetramers, to complete the mature network structure.

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing gamma' chains, which are fibrinogen gamma chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 microM respectively). The presence of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on gamma' chains is a constitutive feature of fibrin as well as fibrinogen.

The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.

Human fibrinogen 1 is homodimeric with respect to its ␥ chains ('␥ A -␥ A '), whereas fibrinogen 2 molecules each contain one ␥ A (␥ A 1-411V) and one ␥ chain, which differ by containing a unique C-terminal sequence from ␥408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the ␥ chains. Second, cross-linking of fibrin ␥ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to ␥ chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to ␥ chains. IntroductionFibrinogen is a multidomain disulfide-linked protein composed of symmetric halves, each consisting of 3 polypeptide chains termed A␣, B␤, and ␥. 1 Human fibrinogen can be separated by ion exchange chromatography into 2 major fractions, fibrinogen 1 (peak 1 fibrinogen) and fibrinogen 2 (peak 2 fibrinogen). 2,3 Plasma fibrinogen contains approximately 15% fibrinogen 2. Structurally, the 2 fibrinogens differ from each other with respect to the composition of their ␥ chains. Fibrinogen 1 contains 2 ␥ A chains, each composed of 411 amino acids, whereas heterodimeric fibrinogen 2 molecules each contain one ␥ A and one ␥Ј chain. 3,4 The variant ␥Ј chain is longer (427 residues) and has a more anionic, carboxyl terminal sequence than the ␥ A chain beyond position 408. 4 Alternative mRNA splicing at the exon 9-exon 10 boundaries gives rise to the variant ␥Ј chain. 5 Thrombin binds to fibrinogen at the substrate site through its exosite 1, 6-8 thereby mediating cleavage of fibrinopeptide A 9-12 and slower cleavage of fibrinopeptide B. 13,14 Fibrin assembly commences with the formation of double-stranded twisting fibrils in which fibrin molecules are arranged in a staggered, overlapping manner. 15 Subsequently, lateral fibril associations occur, resulting in thicker fibrils and fibers. Concomitant with converting fibrinogen to fibrin, thrombin activates factor XIII to factor XIIIa. [16][17][18][19][20][21] In the presence of factor XIIIa and Ca 2ϩ , fibrin undergoes intermolecular covalent cross-linking by the formation of ⑀-amino(␥-glutamyl) lysine isopeptide bonds. 22 Generally speaking, intermolecular cross-linking occurs rapidly between ␥ chains to form ␥-dimers and more slowly among ␣ chains to create oligomers and larger ␣ chain...