Studies on the basis for the properties of fibrin produced from fibrinogen-containing γ′ chains

Abstract

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...

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…68 Their data further suggested that FXIII activation was slower in the presence of ␥A/␥Ј fibrinogen than it was in the presence of ␥A/␥A fibrinogen. 30 FXIII activation peptide cleavage by Atroxin, a snake venom enzyme that cleaves FpA from fibrinogen but does not bind to the ␥Ј chain, was not affected by the presence of ␥A/␥Ј fibrinogen, suggesting that the observed rate differences in the thrombin-catalyzed system were related to thrombin binding to the ␥Ј chain.…”

“…31 It has been suggested that allosteric effects of the binding of the ␥Ј chain to exosite II on the thrombin catalytic site may be responsible for the inhibitory activity of ␥A/␥Ј fibrinogen. 30 Recent reports suggest that the ␥Ј chain may also function as a reservoir for active thrombin in the fibrin clot. Thrombin bound to fibrin was shown to remain functionally active.…”

“…One of these involves inhibition of thrombin activity, which was first reported as antithrombin I more than 50 years ago by Seegers et al 26,27 For comprehensive reviews on this topic, see Mosesson. 28,29 The thrombin clotting time was increased by addition of phosphorylated ␥Ј V408-L427 peptide in a purified system, 30 but the ␥Ј peptide was added at a 80-to 320-fold molar excess over ␥A/␥A fibrinogen, compared with a 0.1 molar ratio in vivo. Reconstitution of fibrinogen into fibrinogen-deficient plasma reduced thrombin generation as measured using a thrombin chromogenic substrate, and ␥A/␥Ј fibrinogen showed more inhibition than ␥A/␥A fibrinogen.…”

Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen γ′ levels

“…58,59 Thrombin interacts with the carboxy terminal sequence of the ␥Ј chain through its exosite 2, 26,27,57 which prevents the participation of this exosite in the binding of thrombin to heparin 60 and which recently was found to be responsible for the observation that thrombin-induced FPA generation was slower with ␥A/␥Ј fibrinogen than with ␥A/␥A fibrinogen. 61 Experiments in which thrombin generation was measured in afibrinogenemia plasma and fibrinogen-depleted plasma supplemented with ␥A/␥A fibrinogen or ␥A/␥Ј fibrinogen showed that ␥A/␥Ј fibrinogen had a more profound effect in down-regulating thrombin generation than ␥A/␥A fibrinogen. 62 Future studies should reveal whether and how a 2-fold reduction of the ␥Ј/␥ ratio would influence fibrin formation and degradation.…”

Reactive carbonyl compounds (RCCs) cause aggregation and dysfunction of fibrinogen

“…In order to clarify whether thrombin can still recognize the cleavage sites in the N-terminal region of fibrinogen to promote fibrinogenesis (Siebenlist et al, 2005), RCC-treated fibrinogen was incubated with thrombin and aliquots were examined by MALDI-TOF mass spectrometry. Cleavage of fibrinogen releases fibrinopeptide A and B, peptides of ∼1900 Da and ∼2400 Da in length.…”