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TC21/RRas2 regulates glycoprotein VI-FcR?-mediated platelet activation and thrombus stability.

ABSTRACT: 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 ?IIb ?3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets. SUMMARY: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-FcR? immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor, in an Src-dependent manner. GPVI-induced platelet aggregation, integrin ?IIb ?3 activation, and ?-granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase C?2, 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-FcR?-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-FcR?-mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

SUBMITTER: Janapati S

PROVIDER: S-EPMC6286703 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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TC21/RRas2 regulates glycoprotein VI-FcRγ-mediated platelet activation and thrombus stability.

Janapati S S Wurtzel J J Dangelmaier C C Manne B K BK Bhavanasi D D Kostyak J C JC Kim S S Holinstat M M Kunapuli S P SP Goldfinger L E LE

Journal of thrombosis and haemostasis : JTH 20180608

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 α<sub>IIb</sub> β<sub>3</sub> integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets.<h4>Summary</h4>Background Many RAS family small GTPases are expres ...[more]

PMID: 29883056

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Project description:Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.

Project description: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.

Dataset Information

TC21/RRas2 regulates glycoprotein VI-FcR?-mediated platelet activation and thrombus stability.

Publications

TC21/RRas2 regulates glycoprotein VI-FcRγ-mediated platelet activation and thrombus stability.

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