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