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Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

ABSTRACT: Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving ?IIb?3.Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-?IIb?3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of ?IIb?3), suggesting that both receptors contribute to thrombus formation on these substrates, but ?IIb?3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

SUBMITTER: Induruwa I

PROVIDER: S-EPMC5838801 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

Induruwa I I Moroi M M Bonna A A Malcor J-D JD Howes J-M JM Warburton E A EA Farndale R W RW Jung S M SM

Journal of thrombosis and haemostasis : JTH 20180115 2

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbβ3.<h4>Summary</h4>Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating throm ...[more]

PMID: 29210180

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Project description:Essentials RAS proteins are expressed in platelets but their functions are largely uncharacterized. TC21/RRas2 is required for glycoprotein VI-induced platelet responses and for thrombus stability in vivo. TC21 regulates platelet aggregation by control of αIIb β3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets. Background Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS-induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored. Objectives To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis. Results We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI-FcRγ immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor, in an Src-dependent manner. GPVI-induced platelet aggregation, integrin αIIb β3 activation, and α-granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal-regulated kinase, were inhibited in TC21-deficient platelets ex vivo. In contrast, these responses were normal in TC21-deficient platelets following stimulation with P2Y, protease-activated receptor 4 and C-type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI-FcRγ-ITAM-specific. TC21 was required for GPVI-induced activation of Rap1b. TC21-deficient mice did not show a significant delay in injury-induced thrombosis as compared with wild-type controls; however, thrombi were unstable. Hemostatic responses showed similar effects. Conclusions TC21 is essential for GPVI-FcRγ-mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

Project description:Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect. SUMMARY:Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through G?q was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the ?IIb ?3 antagonist GR144053, indicating negligible roles for GPVI shedding or ?IIb ?3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

Dataset Information

Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

Publications

Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

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