Conformational Changes in Fragments D and Double-D from Human Fibrin(ogen) upon Binding the Peptide Ligand Gly-His-Arg-Pro-Amide<sup>,</sup>

Everse, Stephen J.; Spraggon, Glen; Veerapandian, L.; Doolittle, Russell F.

doi:10.1021/bi982626w

Cited by 80 publications

(150 citation statements)

References 33 publications

(68 reference statements)

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“…6,15,16 These and subsequent crystal structures also identified the residues that likely form the D:D interface. 16,17 This interface is unusual in 2 ways: it is not symmetric as might be expected for interactions between identical nodules and it is not supported by a significant network of hydrogen bonds or salt links. Nevertheless, specific residues within the ␥ chain appear to be critical to this interface.…”

Section: Introductionmentioning

confidence: 92%

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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Crystallographic structures indicate that ␥-chain residue Asn308 participates in D:D interactions and indeed substitutions of ␥Asn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: ␥Asn308 changed to lysine (␥N308K), isoleucine (␥N308I), and alanine (␥N308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa-catalyzed crosslinking by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of ␥N308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with ␥N308K. Factor XIIIa-catalyzed ␥-␥ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) ␥-␥ dimer formation of only ␥N308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both. IntroductionFibrinogen is a 340-kDa glycoprotein that circulates in the blood at 5.88 to 11.76 M (2-4 g/L). It is composed of a pair of 3 polypeptide chains, A␣, B␤, and ␥, connected by 29 intrachain and interchain disulfide bonds. 1 The 6 chains are arranged into 3 globular nodules. The central E nodule contains the N-termini of all chains and the distal D nodules contain the C-termini of the ␤, ␥, and a short segment of the ␣ chains. Coiled coils composed of all 3 chains link the E and D nodules. 2 The C-terminal residues of A␣ chains form globular nodules that are associated with one another, as well as with the E nodule. 3 Recent crystallographic studies have provided high-resolution structures of the C-terminal region of the ␥ chain, which contains functional sites for fibrin polymerization, calcium binding, platelet aggregation, and factor XIII (FXIII) cross-linking. [4][5][6] During coagulation, thrombin cleaves fibrinogen, releasing fibrinopeptides A (FpA) and B (FpB) from the N-termini of the A␣ and B␤ chains, respectively, and converting fibrinogen to fibrin monomers (for reviews, see Blomback 1 and Doolittle 2 ). Fibrin monomers polymerize spontaneously through a 2-step process, with a second step being calcium-dependent. 7,8 The first step begins with the release of FpA. This exposes the "A" site, which interacts with the "a" site in the D nodule of another molecule, leading to the formation of half-staggered, double-stranded protofibrils. 9,10 Within protofibrils, individual fibrin mol...

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Section: Introductionmentioning

confidence: 92%

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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“…Fibrin(ogen) structure(s) are known from x-ray diffraction studies (12,13,(45)(46)(47)(48)(49), and that of PF4 has been deduced through both x-ray diffraction and NMR spectroscopy (50,51). Three intriguing features of these structures attracted our attention.…”

Section: Pf4 Has a Considerable Potential To Shape Fibrin Molecular Ementioning

confidence: 99%

Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin

Amelot

Tagzirt

Ducouret

et al. 2007

Journal of Biological Chemistry

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Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31-and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 M the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.In addition to catalyzing the conversion of fibrinogen to fibrin, thrombin activates the G protein-coupled protease-activated receptor 1, triggering platelet adhesion and activation (1, 2). Following activation, large quantities of platelet factor-4 (PF4/CXCL4), 3 a major component of the ␣-granules (15-20 g/10 9 platelets), are released into the vicinity of growing blood clots, such that amounts in excess of 5 g/ml are found in serum (3, 4). PF4 is an asymmetrically associated homo-tetrameric (70 residues/subunit) chemokine that interacts with a variant of CXCR3. The physiologic function of PF4 is not fully understood even if one of the first clues to the existence of endogenous angiogenesis inhibitors came with the observation that it inhibits endothelial cell proliferation (5). PF4 is otherwise mainly known for binding polysulfated glycosaminoglycans such as heparin and has been extensively studied for its role in heparin-induced thrombocytopenia (6). Generally speaking, PF4 binds to glycosaminoglycans, modulating their activity. Both pro-coagulant and anti-coagulant properties for PF4 have been reported. PF4 accelerates the activation of the anticoagulant protein C (7, 8); in contrast, studies on transgenic mice demonstrate that whereas PF4 contributes to thrombus formation, PF4-null mice had no overt bleeding diathesis (9).Fibrinogen is a M r 340,000 glycoprotein consisting of a pair of ␣-, ␤-, and ␥-chains. The molecule comprises two outer D-nodules connected through coiled-coil domains to a central E-nodule (10, 11). The E-nodule includes all six NH 2 termini, and each D-nodule contains the COOH terminus of the ␤-and ␥-chains folded into globular domains, namely ␤C and ␥C (12, 13). Fibrin formation is initiated by a thrombin-catalyzed removal of two peptides (fibrinopeptides A) from t...

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“…This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1). The release of FpB, a 14-residue peptide, exposes the "B" site (2)(3)(4), which presumably interacts with a "b" site in the ␤ chain of the D nodule of another molecule (5). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood.…”

mentioning

confidence: 99%

Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

Mullin

Gorkun

Binnie

et al. 2000

Journal of Biological Chemistry

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During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A␤ fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA (G14V). Analyses of fibrinopeptide release from A␤ fibrinogen showed that FpA release and FpA release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA release from A␤ fibrinogen was unaffected. Thus, with A␤ fibrinogen, fibrinopeptide release from the ␤ chain is similar to fibrinopeptide release from the ␣ chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A␤ fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A␤ fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the ␤ chain does not affect polymerization of fibrinogen.Fibrinogen is a 340-kDa plasma protein that is involved in the final phase of the coagulation cascade. Fibrinogen consists of two pairs of three polypeptide chains (A␣, B␤, and ␥) that fold to produce a trinodular protein with two distal (D) nodules connected to a central nodule (E) by coiled-coil regions. The central nodule of the molecule consists of the N termini of all six polypeptide chains, and the D nodules consist predominantly of the C termini of the ␤ and ␥ chains, each folded into a globular domain. To initiate polymerization, the serine protease thrombin cleaves four specific Arg-Gly bonds at the N termini of both the A␣ and B␤ chains, releasing fibrinopeptides A (FpA) 1 and B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the "A" site, which noncovalently interacts with the "a" site in the ␥ chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1). The release of FpB, a 14-residue peptide, exposes the "B" site (2-4), which presumably interacts with a "b" site in the ␤ chain of the D nodule of another molecule (5). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood. The final product of this polymerization is a complex, branching network of fibers.The interactions of thrombin with fibrinogen have been extensively studied (Refs. 6 and 7; for a review, see Ref. 8)....

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Conformational Changes in Fragments D and Double-D from Human Fibrin(ogen) upon Binding the Peptide Ligand Gly-His-Arg-Pro-Amide^,

Cited by 80 publications

References 33 publications

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin

Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

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Conformational Changes in Fragments D and Double-D from Human Fibrin(ogen) upon Binding the Peptide Ligand Gly-His-Arg-Pro-Amide,

Cited by 80 publications

References 33 publications

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin

Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

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Conformational Changes in Fragments D and Double-D from Human Fibrin(ogen) upon Binding the Peptide Ligand Gly-His-Arg-Pro-Amide^,