Role of fibrinogen alpha and gamma chain sites in platelet aggregation.

Farrell, David H.; Thiagarajan, Perumal; Chung, Dominic W.; Davie, Earl W.

doi:10.1073/pnas.89.22.10729

Cited by 318 publications

(224 citation statements)

References 48 publications

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“…The increased clearance of platelets by GoF ADAMTS‐13 may be the result of fibrinogen proteolysis, which removes a αII b β 3 binding site on the fibrinogen Aα chain (Fig. 2C), thereby reducing fibrinogen‐mediated platelet–platelet interactions in the developing thrombus 38. This is supported by the fact that the GPIIbIIIa receptor antagonist GRR144053 induces a similar decrease in platelet coverage (~60%) when perfused over the preformed thrombi in this assay (Fig.…”

Section: Discussionmentioning

confidence: 67%

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

South

Freitas

Lane

2016

Journal of Thrombosis and Haemostasis

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Essentials Recently, ADAMTS‐13 has been shown to undergo substrate induced conformation activation.Conformational quiescence of ADAMTS‐13 may serve to prevent off‐target proteolysis in plasma.Conformationally active ADAMTS‐13 variants are capable of proteolysing the Aα chain of fibrinogen.This should be considered as ADAMTS‐13 variants are developed as potential therapeutic agents. Click to hear Dr Zheng's presentation on structure function and cofactor-dependent regulation of ADAMTS‐13 SummaryBackgroundRecent work has revealed that ADAMTS‐13 circulates in a ‘closed’ conformation, only fully interacting with von Willebrand factor (VWF) following a conformational change. We hypothesized that this conformational quiescence also maintains the substrate specificity of ADAMTS‐13 and that the ‘open’ conformation of the protease might facilitate proteolytic promiscuity.ObjectivesTo identify a novel substrate for a constitutively active gain of function (GoF) ADAMTS‐13 variant (R568K/F592Y/R660K/Y661F/Y665F).MethodsFibrinogen proteolysis was characterized using SDS PAGE and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Fibrin formation was monitored by turbidity measurements and fibrin structure visualized by confocal microscopy.Results ADAMTS‐13 exhibits proteolytic activity against the Aα chain of human fibrinogen, but this is only manifest on its conformational activation. Accordingly, the GoF ADAMTS‐13 variant and truncated variants such as MDTCS exhibit this activity. The cleavage site has been determined by LC‐MS/MS to be Aα chain Lys225‐Met226. Proteolysis of fibrinogen by GoF ADAMTS‐13 impairs fibrin formation in plasma‐based assays, alters clot structure and increases clot permeability. Although GoF ADAMTS‐13 does not appear to proteolyse preformed cross‐linked fibrin, its proteolytic activity against fibrinogen increases the susceptibility of fibrin to tissue‐type plasminogen activator (t‐PA)‐induced lysis by plasmin and increases the fibrin clearance rate more than 8‐fold compared with wild‐type (WT) ADAMTS‐13 (EC50 values of 3.0 ± 1.7 nm and 25.2 ± 9.7 nm, respectively) in in vitro thrombosis models.ConclusionThe ‘closed’ conformation of ADAMTS‐13 restricts its specificity and protects against fibrinogenolysis. Induced substrate promiscuity will be important as ADAMTS‐13 variants are developed as potential therapeutic agents against thrombotic thrombocytopenic purpura (TTP) and other cardiovascular diseases.

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Section: Discussionmentioning

confidence: 67%

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

South

Freitas

Lane

2016

Journal of Thrombosis and Haemostasis

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“…The KQAGDV y-chain sites are at the very ends of the rodlike molecule, whereas the RGD sites are either close to the central domain (RGDF) or positioned near the carboxy-terminal extension of the a-chains (RGDS). The recent observation that RGD -, RGE substitutions did not affect the ability of fibrinogen variants to support platelet aggregation has questioned the relevance of both RGD sites in fibrinogen-aIIbf13 interactions (Farrel et al, 1992). However, in fibrin protofibrils, due to the half-staggered overlap arrangement of the constituent fibrin molecules (Fowler et al, 1981), both the RGDF and KQAGDV sites, coming from different fibrin monomer units, will probably lie in a closer spatial relationship.…”

Section: Discussionmentioning

confidence: 99%

Modeling the α_IIbβ₃ integrin solution conformation

1993

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The (~l I b P 3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of a and fi subunits. Electron microscopic (EM) images of the a[& complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the a r [ b & complex.We have studied the shape of detergent-solubilized a l I b & by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100).Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized ~111bP3 complex, where the relative spatial location of specific domains the size of 5-10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.

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“…Both integrins contain multiple divalent cation-binding motifs that regulate ligand binding. Recent data suggest that platelet aggregation probably requires only the bivalent bridging of fibrinogen between adjacent platelets via y-chain C-termini [39]. In recent years, evidence has been obtained which indicates that the RGD sequences at positions aos cj7 and (I,,, 574 of the fibrinogen molecule probably play only a minor role and neither sequence is required for platelet binding to fibrinogen [13].…”

Section: Discussionmentioning

confidence: 99%

Microenvironmental Changes in Platelet Membranes Induced by the Interaction of Fibrinogen‐Derived Peptide Ligands with Platelet Integrins

Watała

Gwoździński

Pluskota

et al. 1996

European Journal of Biochemistry

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A few studies have confirmed the influence of peptides containing either the RGD or dodecapeptide H-I 2-V (HHLGGAKQAGDV) sequence on cell membrane structure and function. In order to consider previous findings and to explore microenvironmental changes associated with the interaction of these two fibrinogen-derived peptides with platelet membranes, we employed fluorescence quenching and electron paramagnetic resonance techniques to monitor the possible alterations in platelet membrane dynamics induced by RGDS and H-12-V. The interaction of RGDS with platelet membranes resulted in reduced values of the h+,/h,, parameter in both 5-doxylstearic acid and 16-doxylstearic acid spectra indicating a significant rigidification of the membrane lipid bilayer. Otherwise, the fibrinogen-derived peptide that contained the y chain C-terminal sequence H-12-V had a fluidizing effect on the platelet membrane lipid bilayer. The labelling of platelet membranes with 1 -anilino-8-naphthalenesulphonate (ANS) enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and ANS embedded into the membrane lipid bilayer. As RGDS interacts with platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. The opposite alterations were recorded upon interaction of platelet membranes with H-I 2-V. Furthermore, a small shift towards longer wavelengths, which accompanies the spectra of ANS in control platelet membranes, vanishes during the interaction with the peptide H-12-V. This observation can be accounted for by a decrease in the polarity of the ANS environment, and may suggest an enhanced contact of the membrane tryptophan with phospholipid fatty acids. Thus, the data indicate that after the action of H-I 2-V on platelet membrane receptors, the membrane tryptophan residues become exposed to the external environment and the quenchable fraction of membrane tryptophan becomes smaller. The increase (a) in the relative rotational correlation time (z,) of 4-(ethoxyfluorophosphinyloxy)-2,2,6,6-tetramethylpiperidine-I -oxyl (ethoxyfluorophosphinyloxy-TEMPO) and (b) in the hJh, ratio in the spectra of 4-maleimido-2,2,6,6-tctramethylpiperidine-l -oxyl (maleimido-TEMPO) indicate that under these conditions there is an effective immobilization of some domains located on the hydrated surface of membrane proteins and mobilization of those domains buried inside the membrane protein molecules. The interaction of RGDS with platelet membrane integrins resulted in contrary effects, as compared to H-12-V. In conclusion, our spectroscopic data indicate that these two fibrinogen-derived peptides induce opposite effects in the dynamics of platelet membrane components. hexapeptide Gly-Arg-Gly-Glu-Ser-Pro; H-12-V, dodecapeptidc His-HisLeu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-~~1; h , ,/h,,, the ratio of lowfield to mid-field line heights in 5/16-DOXYL-Ste spectra; 12, and /I,, low-field line heights corresponding to weakly and strongly immobilized male...

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Role of fibrinogen alpha and gamma chain sites in platelet aggregation.

Cited by 318 publications

References 48 publications

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Modeling the α_IIbβ₃ integrin solution conformation

Microenvironmental Changes in Platelet Membranes Induced by the Interaction of Fibrinogen‐Derived Peptide Ligands with Platelet Integrins

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Role of fibrinogen alpha and gamma chain sites in platelet aggregation.

Cited by 318 publications

References 48 publications

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

Modeling the αIIbβ3 integrin solution conformation

Microenvironmental Changes in Platelet Membranes Induced by the Interaction of Fibrinogen‐Derived Peptide Ligands with Platelet Integrins

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Modeling the α_IIbβ₃ integrin solution conformation