Crystal Structure of Fragment Double-D from Human Fibrin with Two Different Bound Ligands

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

doi:10.1021/bi985060k

Cited by 66 publications

(128 citation statements)

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“…[22][23][24][25] Each E A site subsequently combines with a constitutive complementary binding pocket (Da) in the D domain of neighboring molecules that is located between γ 337 and γ 379. [26][27][28] These initial E A :Da associations result in formation of double-stranded twisting fibrils in which fibrin molecules become aligned in an end-to-middle staggered overlapping domain arrangement (see F IGURE 2). [29][30][31] Fibrils undergo lateral associations and form branches that result in a complex fiber network.…”

Section: Fibrinogen Conversion To Fibrin and Fibrin Assemblymentioning

confidence: 99%

“…21 E B is utilized through interactions with a constitutive complementary D b site located in the C-terminal β chain segment of the D domain. 28,42,43 The E B :D b interaction is not absolutely required for lateral fibril and fiber associations, but it contributes to this process through cooperative interactions resulting from alignment of D domains in the assembling polymer. 42 Polymerization of des BB-fibrin results in the same type of fibril structure as occurs with des AA-fibrin, 38 but the clot strength is lower than that of des AA-fibrin.…”

Section: Molecular and Cellular Interactions Following Fibrinopeptidementioning

confidence: 99%

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The Structure and Biological Features of Fibrinogen and Fibrin

Mosesson

Siebenlist

Meh

2001

Annals of the New York Academy of Sciences

559

349

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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.

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Section: Fibrinogen Conversion To Fibrin and Fibrin Assemblymentioning

confidence: 99%

Section: Molecular and Cellular Interactions Following Fibrinopeptidementioning

confidence: 99%

The Structure and Biological Features of Fibrinogen and Fibrin

Mosesson

Siebenlist

Meh

2001

Annals of the New York Academy of Sciences

559

349

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“…FpA release is the key initial event, with A-a interactions governing (proto)fibril formation in a final half-staggered, double-stranded arrangement. 20,21 FpB is released by thrombin later in the process, and the B-b engagement enhances the lateral thickening of the fibers. 22,23 There is also evidence of promiscuity between the A and B knobs toward the a and b holes, probably derived from the common evolutionary origin of the fibrinogen chains.…”

Section: Introductionmentioning

confidence: 99%

Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a “Double‐Detroit” recombinant fibrinogen mutant and knobs‐mimic peptides

Duval

Aprile

Salis³

et al. 2020

Journal of Thrombosis and Haemostasis

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Background Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α‐ and β‐chains N‐termini (GPR, GHR; A‐, B‐knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ‐ and (B)β‐chains C‐terminal regions. A role for mostly disordered (A)α‐chains C‐terminal regions “bridging” between fibrin molecules/fibrils has been proposed. Objectives Fibrinogen Detroit is a clinically observed mutation (AαR19 → S) with nonengaging GPS A‐knobs. By analogy, a similar Bβ‐chain mutation, BβR17 → S, should produce nonengaging GHS B‐knobs. A homozygous “Double‐Detroit” mutant (AαR19 → S, BβR17 → S; DD‐FG) was developed: with A‐a and B‐b engagements endogenously blocked, other interactions would become apparent. Methods DD‐FG, wild‐type recombinant (WT‐FG), and human plasma (hp‐FG) fibrinogen self‐association was studied by turbidimetry coupled with fibrinopeptides release high‐performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light‐scattering following size‐exclusion chromatography (SE‐HPLC). Results In contrast to WT‐FG and hp‐FG, DD‐FG produced no turbidity increase, irrespective of thrombin concentration. The SE‐HPLC profile of concentrated DD‐FG was unaffected by thrombin treatment, and light‐scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp‐FG, both WT‐FG and DD‐FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS‐PAGE/Western‐blots revealed partial Bβ‐chain N‐terminal and Aα‐chain C‐terminal degradation. Nevertheless, ~70% DD‐FG molecules bearing (A)αC‐regions potentially able to associate were available. Higher‐concentration, nearly intact hp‐FG with 500‐fold molar excess GPRP‐NH2/GHRP‐NH2 knobs‐mimics experiments confirmed these no‐association findings. Conclusions (A)αC‐regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.

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“…In the construction of the model, we have taken into consideration the fact that site 'B' (Bb15-18 belonging to another strand within the protofibril. After fibrinopeptide B is split off the site, 'B' is formed, and the latter interacts intermolecularly with the complementary site 'b' situated in the bC-domain in a 'knob'-'hole' type of interaction [7,9]. The remaining part of site 'C' (Bb19-46) probably remains bound to the fibrin D-domain.…”

Section: Discussionmentioning

confidence: 99%

Functional role of Bβ‐chain N‐terminal fragment in the fibrin polymerization process

et al. 2007

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Fibrinogen is a central protein of the blood coagulation system. The human fibrinogen molecule (molecular mass 344 kDa) consists of two identical subunits connected by disulfide bonds [1]. Each monomer subunit is formed by three nonidentical polypeptide chains, Aa, Bb and c. The N-terminal ends of all six polypeptide chains are situated in the fibrinogen central region, which is known as the E-domain. Two Four mAbs of the IgG 1 class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bb15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bb26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fabfragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bb15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbSARGHRPLDKKREEA(12-26), , , and , which imitate the various sequences in the N-terminal region of the fibrin Bb-chain, have been investigated. Peptides Bb12-26 and Bb26-46, but not Bb40-46, Bb19-26, and Bb26-36, proved to be specific inhibitors of fibrin polymerization. The IC 50 values for Bb12-26 and Bb26-46 were 2.03 · 10 )4 and 2.19 · 10 )4 m, respectively. Turbidity and electron microscopy data showed that peptides Bb12-26 and Bb26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bb12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aa17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bb12-46 of one fibrin desAA molecule and the D-domain of another has been constructed. ; NaCl ⁄ P i , 0.02 M potassium phosphate buffer (pH 7.4) with 0.13 M NaCl; t-NDSK, thrombin-treated N-terminal disulfide knot of fibrin; TPBS, NaCl ⁄ P i with 0.05% Tween-20.

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Crystal Structure of Fragment Double-D from Human Fibrin with Two Different Bound Ligands

Cited by 66 publications

References 0 publications

The Structure and Biological Features of Fibrinogen and Fibrin

The Structure and Biological Features of Fibrinogen and Fibrin

Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a “Double‐Detroit” recombinant fibrinogen mutant and knobs‐mimic peptides

Functional role of Bβ‐chain N‐terminal fragment in the fibrin polymerization process

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