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Collagen Promotes Sustained Glycoprotein VI Signaling in Platelets and Cell Lines

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Collagen Promotes Sustained Glycoprotein VI Signaling in Platelets and Cell Lines Journal of Thrombosis and Haemostasis, 5: 2274–2283 ORIGINAL ARTICLE Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines M. G. TOMLINSON,* S. D. CALAMINUS,* O. BERLANGA,* J. M. AUGER,* T. BORI-SANZ,* L. MEYAARD andS. P. WATSON* *Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK; and Department of Immunology, University Medical Center, Utrecht, The Netherlands To cite this article: Tomlinson MG, Calaminus SD, Berlanga O, Auger JM, Bori-Sanz T, Meyaard L, Watson SP. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines. J Thromb Haemost 2007; 5: 2274–83. a cell line NFAT assay will facilitate the molecular dissection of Summary. Background: Glycoprotein (GP)VI is the major GPVI signaling and the identification of GPVI antagonists in signaling receptor for collagen on platelets and signals via the drug discovery. associated FcRc-chain, which has an immunoreceptor tyrosine- containing activation motif (ITAM). Objective: To determine Keywords: collagen, convulxin, glycoprotein VI, leukocyte- why GPVI–FcRc signals poorly, or not at all, in response to associated immunoglobulin-like receptor-1, platelets, signaling. collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and Introduction results: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained Platelets play an essential role in hemostasis by supporting ITAM signaling, we demonstrate collagen-induced GPVI– clot formation at sites of vascular injury. However, platelet FcRc signaling in hematopoietic cell lines. This is accompanied activation in diseased arteries can give rise to thrombotic by relatively weak but sustained protein tyrosine phosphoryla- diseases such as myocardial infarction and stroke. Extracel- tion, in contrast to the stronger but transient response to lular matrix proteins play a critical role in initiating platelet convulxin. Sustained signaling by collagen is also observed in adhesion and platelet activation as well as securing the platelets and is necessary for the maintenance of spreading on thrombus at the site of injury. Collagen is the most collagen. Finally, in cell lines, the inhibitory collagen receptor thrombogenic component of the subendothelial matrix and leukocyte-associated immunoglobulin-like receptor-1 (LAIR- is thought to induce powerful platelet activation by a Ôtwo- 1), which is not expressed on platelets but is present on most site, two-stepÕ model[1].Inthismodel,theinitialeventis hematopoietic cells, inhibits GPVI responses to collagen but not collagen binding to its major signaling receptor, the immu- convulxin. Conclusion: The inability of previous studies to noglobulin superfamily protein glycoprotein (GP)VI–FcRc- readily detect GPVI collagen signaling in cell lines is probably chain complex, and activation of a tyrosine kinase cascade because of the weak but sustained nature of the signal and the downstream of the FcRc immunoreceptor tyrosine-based presence of the inhibitory collagen receptor LAIR-1. In activation motif (ITAM). This generates a relatively weak platelets, we propose that GPVI–FcRc has evolved to transmit GPVI–FcRc signal that results in Ôinside-outÕ activation of sustained signals in order to maintain spreading over several the major adhesive receptor for collagen, the integrin a2b1, hours, as well as facilitating rapid activation through release of and release of the secondary mediators ADP and throm- feedback agonists and integrin activation. The establishment of boxane A2 (TXA2). These secondary mediators feed back on the G-protein-coupled receptors P2Y1/P2Y12 and TXA2R, respectively, which further activate a2b1. The promotion of collagen binding to activated a2b1 brings about a net increase in collagen–GPVI interactions and robust signaling [1]. Correspondence: Michael G. Tomlinson, Centre for Cardiovascular Despite the widespread acceptance of GPVI as a key Sciences, Division of Medical Sciences, Institute of Biomedical signaling molecule in this scheme, it has proven difficult to Research, Wolfson Drive, University of Birmingham, Birmingham B15 2TT, UK. observe activation by collagen in GPVI-transfected cell lines, Tel.: +44 121 414 8308; fax: +44 121 415 8817; e-mail: in contrast to the robust response to the snake venom toxin [email protected] convulxin [2–5]. A potential explanation is that GPVI collagen signaling is weak and therefore difficult to detect Received 22 March 2007, accepted 14 August 2007 in cell lines in the absence of positive feedback activation or Ó 2007 International Society on Thrombosis and Haemostasis Collagen promotes sustained GPVI signaling 2275 activated a b . Indeed, several studies have documented the 2 1 Materials and methods weak nature of the response to collagen in platelets in the presence of inhibitors of ADP and TXA ,orblockadeof 2 Cells, antibodies and GPVI agonists a2b1 [6,7]. In contrast to this idea, Chen et al. [8] have proposed that the inability of transfected cell lines to support The RBL-2H3 rat basophilic leukemia cell line was cultured in signaling responses to collagen results from the need for a DulbeccoÕs modified EagleÕs medium supplemented with 10% critical level of expression of GPVI. The evidence in support fetal bovine serum, penicillin, streptomycin, and glutamine. of this is the demonstration of reconstitution of collagen RBL-2H3 cells that stably expressed human GPVI were signaling in a single stably transfected GPVI-expressing RBL- generated as previously described [14]. Wild-type DT40 chicken 2H3 cell line that had at least a 2-fold greater level of B-cells and lines deficient for Lyn [15], Btk [16], Syk [15] and expression of GPVI than other, unresponsive stably trans- PLCc2 [17] were cultured in RPMI supplemented with 10% fected RBL-2H3 cell lines. This cannot be the full explana- fetal bovine serum, 1% chicken serum, 50 lM 2b-mercapto- tion, however, as platelets express a similar level of GPVI to ethanol, penicillin, streptomycin, and glutamine. Human that reported in this study [8] and yet are still able to respond washed platelets were prepared as previously described [18]. to collagen if the GPVI level is reduced by 5-fold to fiftyfold, The mouse anti-human GPVI monoclonal antibody (mAb) even in the presence of blockade of a2b1 [9,10]. 204-11 was as previously described [19], the mouse anti- In the present study, we have investigated the hypothesis phosphotyrosine mAb 4G10 was from Upstate (Charlottes- that GPVI does not promote marked collagen signaling in ville, VA, USA), and the anti-human a2mAb6F1wasagift cell lines because of the weak nature of the response and the from Barry Coller (Rockefeller University, New York, NY, absence of feedback agonists. Accordingly, we have moni- USA). Collagen (Horm) was from Nycomed (Munich, Ger- tored nuclear factor of activated T-cells (NFAT)–luciferase many), collagen-related peptide (CRP) was prepared as transcriptional reporter activity, which is a highly sensitive described previously [20], and the snake venom toxin convulxin readout and is widely used to study signaling through the was a gift from M. Leduc and C. Bon (Unite des Venens, ITAM-containing B-cell and T-cell receptors. The NFAT Institut Pasteur, Paris, France). reporter contains three copies of a composite NFAT– activator protein-1 (AP-1) element from the human interleu- Plasmids kin-2 (IL-2) gene promoter [11], and is maximally activated by combined Ca2+ elevation and RAS/mitogen-activated The GPVI expression construct, containing a MYC epitope protein kinase (MAPK) signaling, which activate NFAT and tag at the C-terminus, was generated by polymerase chain AP-1, respectively. In resting cells, NFAT proteins are reaction (PCR), using human GPVI cDNA [14] as a template, maintained in the cytoplasm in an inactive, phosphorylated and cloned into pRC (Invitrogen, Paisley, UK). The FcRc- state, but upon Ca2+ elevation, the serine–threonine phos- chain expression construct was generated by PCR, using phatase calcineurin is activated, resulting in dephosphoryla- human erythroleukemia cell line cDNA as a template, and tion of NFAT, exposure of its nuclear localization signal, cloned into pEF6 (Invitrogen). The pCDNA3.1–LAIR-1– and translocation to the nucleus [12]. Sustained calcineurin CD3f chimeric construct, expressing the extracellular region of activation is required to maintain NFAT dephosphorylation human LAIR-1 and the transmembrane and intracellular and nuclear localization, and this is efficiently achieved with regions of human CD3f [21], and the pCDNA3.1–LAIR-1 even very low continuous Ca2+ elevations that are only construct, expressing human LAIR-1 [22], were generated as slightly above baseline levels [13]. Therefore, NFAT-driven previously described. The NFAT–luciferase reporter contained luciferase generation over a period of several hours is a three copies of the distal NFAT site from the IL-2 promoter highly sensitive readout for weak, sustained signaling that [11]. The pEF6–lacZ expression construct was from Invitrogen. may not be detectable by, for example, increases in intracellular Ca2+. Transfections and luciferase assays Our results demonstrate that collagen does indeed pro- mote weak but sustained GPVI signaling, resulting in RBL-2H3 and DT40 cells were transfected in a volume of marked NFAT activation in RBL-2H3 mast and DT40 0.4 mL of cytomix buffer (120
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