Journal of Thrombosis and Haemostasis, 5: 2274–2283

ORIGINAL ARTICLE promotes sustained glycoprotein VI signaling in 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 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 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, 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

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 mM KCl, 0.5 mM CaCl2,10mM B-cell lines. Collagen signaling is particularly strong in DT40 K2HPO4,10mM KH2PO4,25mM HEPES, 2 mM EGTA, cells, which we propose is due in part to the absence of an 5mM MgCl2, pH 7.6) using a Gene Pulser Electroporator (Bio- inhibitory collagen receptor, leukocyte-associated immuno- Rad, Hercules, CA, USA) set at 276 V and 950 lF(RBL-2H3) globulin-like receptor-1 (LAIR-1), which is expressed by or 350 V and 500 lF (DT40). For luciferase assays, RBL-2H3 most hematopoietic cell lines, with the exception of platelets and DT40 cells were transfected with the expression constructs and B-cell lines. We further demonstrate sustained collagen of interest, 20 lg of NFAT–luciferase reporter construct and signaling through GPVI in platelets, and suggest that 2 lg of pEF6–lacZ to control for transfection efficiency. Sixteen sustained signals via this receptor are required to maintain hours after transfection, live cells were counted by Trypan blue platelet spreading on collagen and thereby contribute to exclusion, and samples were divided for luciferase assay, thrombus stability. b-galactosidase assay, and flow cytometry, as previously

2007 International Society on Thrombosis and Haemostasis 2276 M. G. Tomlinson et al described [23]. Luciferase assay data were normalized to b- optics with a Zeiss 63 · oil immersion 1.40 NA plan-apochro- galactosidase values or expressed as the percentage of the mat lens on a Zeiss Axiovert 200M microscope. Digital images positive control response to phorbol 12-myristate 13-acetate were captured by a Hamamatsu Orca 285 cooled digital camera (PMA) and ionomycin. GPVI expression levels on transfected using SLIDEBOOK 4.0 (Intelligent Imaging Innovations, Inc., cells were detected by staining with 10 lgmL)1 of anti-human Denver, CO, USA). The degree of platelet adhesion and GPVI mAb, followed by fluorescein isothiocyanate-conjugated surface area of spread platelets were quantified as previously anti-mouse secondary antibody, and analyzed using a FAC- described [24]. Scalibur (BD Biosciences, Oxford, UK) in combination with CELLQUEST software. Results

Ca2+ fluorimetry Collagen induces relatively weak but sustained phosphotyrosine signaling in GPVI-transfected RBL-2H3 cells Ca2+ mobilization in RBL-2H3 cells was measured by fluorimetry, using the Fura-2 Ca2+ reporter dye, as previously Previous reports have shown that collagen-induced Ca2+ described [14]. mobilization and early tyrosine phosphorylation is relatively weak or undetectable in GPVI-transfected RBL-2H3 mast cells [4,8], Jurkat T-cells [5] and DAMI megakaryocyte-like cells [2], Protein tyrosine phosphorylation despite robust responses to convulxin. In line with this, we also Transfectedcelllineswereincubatedfor30mininRPMIat failed to observe collagen-induced Ca2+ signaling in RBL-2H3 37 C before stimulation. Cell line concentrations were cell stable GPVI transfectants, in contrast to the robust ) ) 1 · 107 mL 1 for RBL-2H3 and 4 · 107 mL 1 for DT40. response to convulxin (Fig. 1A). This led us to hypothesize that ) Human washed platelets (5 · 108 mL 1) were preincubated for the collagen-induced GPVI signal might be more readily 10mininTyrodeÕs buffer [18] at 37 Cwith1mM EGTA, detected by assaying over a longer time course, based on the )1 )1 2UmL apyrase, 10 lM indomethacin and 10 lgmL 6F1 prediction that the signal is weak and delayed. To address this mAb. After stimulation with collagen (30 lgmL)1) or con- issue, cells were stimulated over a 60-min time course, and vulxin (10 lgmL)1), cell lines and platelets were lyzed at the signaling responses were detected by anti-phosphotyrosine appropriate time point by pipetting into an equal volume of ice- blotting of whole cell lysates. Collagen stimulated weak cold 2 · NP40/dodecyl maltoside lysis buffer (1.6% NP40, 2% tyrosine phosphorylation of a limited number of proteins after dodecyl maltoside, 20 mM Tris, pH 7.5, 300 mM NaCl, 4 mM a delay, whereas the convulxin response was strong and rapid )1 EDTA, 2 mM dithiothreitol, 400 lgmL 4-(2-aminoethyl) (Fig. 1B; major increases in tyrosine phosphorylation are benzenesulfonyl fluoride (AEBSF), 20 lgmL)1 aprotinin, indicated by asterisks). However, the stimulation of tyrosine )1 )1 20 lgmL leupeptin, 2 lgmL pepstatin, 20 mM NaF and phosphorylation by convulxin returned to almost resting levels 4mM sodium orthovanadate). Insoluble material was removed by 30 min, indicating marked desensitization, whereas the by centrifugation, and samples were separated by sodium small increase induced by collagen was sustained for 60 min dodecylsulfate polyacrylamide gel electrophoresis on 4–12% (Fig. 1B). These data show that convulxin promotes robust gels (Invitrogen) and transferred to a poly(vinylidene difluo- phosphotyrosine signaling through GPVI that is rapid and ride) membrane. Anti-phosphotyrosine western blotting was transient, whereas collagen promotes relatively weak but detected using Western Lightning chemiluminescence reagents sustained phosphotyrosine signaling. (Perkin Elmer, Boston, MA, USA) and Hyperfilm (Amersham Biosciences, Buckinghamshire, UK), which was developed Collagen induces NFAT activation in GPVI-transfected RBL- using a Compact X4 film processor (Xograph Imaging 2H3 cells Systems, Gloucestershire, UK). Activation of the NFAT transcription factor can be used to detect weak but sustained signaling events that include Ca2+ Platelet spreading signals that are undetectable by fluorimetry [13]. This suggests Coverslips were coated with collagen (100 lgmL)1), and that NFAT activation might be a suitable assay for detection of platelet spreading assays were performed as previously GPVI activation by collagen in RBL-2H3 cells, in light of the described [24]. For the present study, human washed platelets undetectable Ca2+ mobilization and weak but sustained ) (2 · 107 mL 1) were allowed to spread on collagen at 37 Cin phosphotyrosine response. To investigate this, RBL-2H3 cells )1 the presence of apyrase (2 U mL ), indomethacin (10 lM)and were transiently transfected with GPVI and FcRc, together integrilin (9 lM). For real-time spreading analysis, images were with a luciferase reporter construct under the control of taken by differential interference contrast (DIC) microscopy at NFAT–AP-1 sites from the IL-2 promoter [11]. This composite 5-s intervals. At 15 min, dimethylsulfoxide vehicle control or NFAT promoter requires both Ca2+ and MAPK signals for PP1 (20 lM) was added, and the morphology of fully spread activation. A b-galactosidase construct was cotransfected to platelets continued to be monitored for a further 15 min. control for transfection efficiency. The transient transfection of Platelets were imaged using Ko¨hler illuminated Nomarski DIC multiple constructs into the same cell is an attractive and

2007 International Society on Thrombosis and Haemostasis Collagen promotes sustained GPVI signaling 2277

A Fig. 1. Collagen induces relatively weak but sustained in glycoprotein (GP)VI–FcRc-transfected RBL-2H3 mast cells. (A) RBL- Collagen Convulxin 2H3 cells stably transfected with GPVI were stimulated with collagen Ca2+ ) ) (30 lgmL 1) or convulxin (10 lgmL 1), and Ca2+ mobilization was measured by fluorimetry using the Ca2+ reporter dye Fura-2. These data arerepresentativeofsixindependent clones. (B) RBL-2H3 cells stably transfectedwithGPVIwerestimulatedwithcollagen(30lgmL)1)or 40 s convulxin (10 lgmL)1), and lyzed at the indicated time points, and pro- teins were separated by sodium dodecylsulfate polyacrylamide gel elec- B Collagen Convulxin trophoresis and western blotted using the anti-phosphotyrosine 0 1' 10' 60' 01'10'60' monoclonal antibody 4G10. Asterisks indicate tyrosine phosphorylation 20'' 3' 30' 20'' 3' 30' that is sustained for collagen or transient for convulxin stimulation. The 175 175 results are representative of three experiments. (C) The RBL-2H3 cell line * was transfected with a nuclear factor of activated T-cells (NFAT)–lucif- 83 * 83 * erase reporter construct, a b-galactosidase construct driven by the 62 62 elongation factor (EF)-1a promoter to control for transfection efficiency, 48 48 * * and 2 lgeachofGPVIandFcRc expression constructs or empty vector 33 33 25 * controls. Sixteen hours post-transfection, expression of GPVI was 17 25 7 17 confirmed by flow cytometry (data not shown). Cells were either left 7 unstimulated, or were stimulated with collagen (10 lgmL)1), collagen- C related peptide (10 lgmL)1), convulxin (10 lgmL)1)orPMA )1 80 (50ngmL ) plus ionomycin (1 lM). Six hours later, cells were lyzed and assayed for luciferase and b-galactosidase. Luciferase data were normal- 60 ized for b-galactosidase values. Error bars represent the standard error of Control the mean from three independent experiments. (D) RBL-2H3 cells were 40 GPVI/FcRγ activity transfected with an NFAT–luciferase reporter construct, a b-galactosidase 20 expression construct to control for transfection efficiency and 0, 0.08, 0.4 or 2 lgeachofGPVIandFcRc expression constructs. GPVI expression

Relative NFAT-luciferase Relative 0 was detected by flow cytometry, and geometric mean fluorescence intensities of GPVI-positive cells were 0, 1.3, 8.7 and 20.1 relative fluo-

CRP rescence units for 0, 0.08, 0.4 and 2 lgoftransfectedDNA, respectively PMA + Collagen (data not shown). Cells were stimulated, and NFAT–luciferase assays were Convulxin ionomycin performed as described in (C). The luciferase data are presented as a Unstimulated percentage of the positive control PMA plus ionomycin response. The D Collagen Convulxin control b-galactosidase levels were similar for each transfection, suggesting 15 60 similar transfection efficiencies (data not shown). Error bars represent the standard error of the mean from three independent experiments. 10 40

20 5 consistent with the proposal that collagen transmits a sustained 0 0 signal through GPVI. 0 0.080.4220 0.08 0.4 It has been reported that elevation of Ca2+ by collagen in NFAT-luciferase activity NFAT-luciferase Quantity of GPVI and FcRγ transfected (µg) (percent PMA + ionomycin) RBL-2H3 cells requires a level of GPVI expression of the same order as that found on platelets [8]. We therefore investigated whether collagen signaling using the NFAT reporter assay also well-established feature of this assay, because data can be requires a threshold level of GPVI. To examine this, RBL-2H3 rapidly obtained without the need to generate stable cell lines. cells were transfected with 0, 0.08, 0.4 or 2 lgofGPVIand With this assay, collagen stimulated an approximately tenfold FcRc constructs. The degree of NFAT activation correlated increase in NFAT–luciferase activation in GPVI-transfected with the quantity of transfected GPVI–FcRc for both collagen but not mock-transfected RBL-2H3 cells over a time period of and convulxin stimulation (Fig. 1D), with no apparent thresh- 6 h (Fig. 1C). CRP, a synthetic for GPVI, similarly old of expression as described for Ca2+ elevation [8]. Therefore activated NFAT, suggesting that collagen-induced NFAT we conclude that GPVI responses to collagen, as detected by activation is not enhanced by an endogenous collagen receptor NFAT activation, do not require a threshold level of GPVI. such as a collagen-binding integrin. In comparison, convulxin stimulated an approximately 3-fold greater response than that Investigation of collagen signaling through GPVI in DT40 B- for collagen. The phorbol ester PMA and the Ca2+ ionophore cells ionomycin, which together activate NFAT downstream of GPVI via protein kinase C/RasGRP and Ca2+, respectively, One of the limitations of the RBL-2H3 cell line in investigating induced similar NFAT activation in both control and GPVI– GPVI signaling is the endogenous expression of the FcRc chain FcRc-transfected cells (Fig. 1C). These results demonstrate as part of FceRI, the high-affinity receptor for IgE. As such, that the NFAT–luciferase assay is a novel and robust readout transfected GPVI may exist, at least in part, as a component of for collagen-induced GPVI signaling in cell line models and are the FceRI complex in RBL-2H3 cells. Consistent with this idea,

2007 International Society on Thrombosis and Haemostasis 2278 M. G. Tomlinson et al transfected GPVI is detected at the cell surface of RBL-2H3 fected but not mock-transfected cells (Fig. 2A). Strikingly, the cells in the absence of FcRc transfection [4,14]. We therefore collagen response (approximately seven hundredfold over sought to identify an FcRc-deficient alternative to RBL-2H3, basal) was significantly greater than that for both convulxin in which GPVI cannot be expressed at the cell surface without (approximately fortyfold) and the positive control PMA and FcRc, as is the case in platelets [25]. The DT40 B-cell line, the ionomycin (Fig. 2A). This is unlikely to be due to synergy with most widely used model system for the study of B-cell receptor an endogenous collagen receptor, because CRP induction of signaling, because of the relative ease with which genes can be NFAT (approximately three hundred and fiftyfold over basal) deleted by homologous recombination [26], was found to was also substantially greater than for convulxin (Fig. 2A). satisfy this requirement, as robust GPVI expression was only This is also not due to different kinetics of NFAT activation, as detected at the cell surface when it was cotransfected with FcRc the collagen response was substantially stronger than the (data not shown). convulxin response, whether measured after 3, 4.5 or 6 h (data The NFAT–luciferase assay was employed in GPVI–FcRc- not shown). The collagen response was concentration-depen- transfected DT40 cells to investigate the ability of this cell line dent and highly sensitive, with hundredfold NFAT activation to reconstitute responses to collagen. Collagen, CRP and detectable at a collagen concentration of 0.1 lgmL)1 convulxinstimulatedNFATactivationinGPVI–FcRc-trans- (Fig. 2B), which is subthreshold for platelet activation.

AB Control 800 GPVI/FcRγ 800

600 600

400 400

200 200 0 0 0 0.1 1 10 Relative NFAT-Iuciferase activity Relative NFAT-Iuciferase activity Collagen (µg mL–1) CRP

PMA + D Collagen Convulxin Collagen Convulxin ionomycin 0 1' 10' 60' 0 1' 10' 60' Unstimulated 20'' 3' 30' 20'' 3' 30' C 150 175 * 125 * 83 * 100 Blot: 62 * pTyr 75 48 33 50 25 17 25 7 NFAT-Iuciferase activity

(percent PMA + ionomycin) 0 83 Collagen: –+ –+ –+ –+ Blot: WT Btk– Syk– PLCγ2– 62 GPVI

Fig. 2. Collagen induces relatively strong nuclear factor of activated T-cells (NFAT) activation and sustained tyrosine phosphorylation in glycoprotein (GP)VI–FcRc-transfected DT40 B-cells. (A) The DT40 B-cell line was transfectedwithanNFAT–luciferase reporter construct, a b-galactosidase construct to control for transfection efficiency, and 2 lgeachofGPVIandFcRc expression constructs or empty vector controls. Sixteen hours post-transfection, expression of GPVI was confirmed by flow cytometry (data not shown). Cells were stimulated, and NFAT–luciferase assays were performed as described in the legend to Fig. 1C. Luciferase data were normalized for b-galactosidase values. Error bars represent the standard error of the mean from three independent experiments. (B) NFAT–luciferase reporter assays were performed on GPVI–FcRc-transfected DT40 cells as described above, but using 0, 0.1, 1 and 10 lgmL)1 collagen as the stimuli. Error bars represent the standard error of the mean from three independent experiments. (C) Wild-type (WT), Btk-deficient (Btk)), Syk-deficient (Syk))andPLCc2-deficient (PLCc2)) DT40 cells were transfected with GPVI, FcRc and NFAT–luciferase constructs, and stimulated with collagen or PMA and ionomycin; luciferase activity was then detected as described in Fig. 1C. The luciferase data are presented as a percentage of the positive control PMA plus ionomycin responses, which were robust for each mutant cell line (between two hundredfold and eight hundredfold over basal). Error bars represent the standard error of the mean from three independent experiments. Transfected GPVI levels were similar for each cell line, as detected by flow cytometry (data not shown). (D) DT40 cells were transfected with 20 lgeachofGPVIandFcRc expression constructs. The cells were stimulated with collagen (30 lgmL)1)orconvulxin(10lgmL)1), and lyzed at the indicated time points, and proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and western blotted using the anti-phosphotyrosine monoclonal antibody (mAb) 4G10. Asterisks indicate tyrosine phosphorylation that is sustained for collagen or transient for convulxin stimulation (upper panels). The blots were stripped and reprobed with the anti-myc mAb 9B11 to detect myc-tagged GPVI (lower panels). The results are representative of three experiments.

2007 International Society on Thrombosis and Haemostasis Collagen promotes sustained GPVI signaling 2279

Collagen induction of NFAT was entirely dependent on existence of a negative regulatory collagen receptor on RBL- PLCc2 and the tyrosine kinase Syk, and was largely, although 2H3 but not DT40 cells. A candidate is the recently identified not completely, dependent on the tyrosine kinase Btk (Fig. 2C). collagen receptor LAIR-1 [21], which contains two immuno- This mimics GPVI signaling requirements in platelets [27], receptor tyrosine-based inhibitory motifs (ITIMs) and can indicating that collagen is signaling through the same pathway negatively regulate ITAM signaling [31,32]. Moreover, LAIR-1 in the transfected cell line. is expressed in most hematopoietic cells, including RBL-2H3 The observed robust collagen-induced NFAT activation is cells as detected by reverse transcription PCR (L. Meyaard, likely to reflect sustained tyrosine kinase signaling. As was the unpubl. data), but is not expressed on most B-cell lines or case for RBL-2H3 cells, convulxin stimulated a rapid but platelets [31,33]. To first test whether LAIR-1 binds to collagen transient increase in DT40 cell tyrosine phosphorylation which but not the structurally unrelated convulxin, NFAT assays returned to resting levels within 30 min, whereas the response were performed using a LAIR-1–CD3f chimeric construct, to collagen was delayed but sustained (Fig. 2D, upper panels; containing the LAIR-1 extracellular domain and the trans- major increases in tyrosine phosphorylation are indicated by membrane and ITAM-containing cytoplasmic regions of asterisks) and of a greater relative magnitude than that CD3f, which activates NFAT in response to collagen [21]. In observed in RBL-2H3 cells (compare Figs 1B and 2D). DT40 cells, both LAIR-1–CD3f and GPVI–FcRc induced Importantly, the difference in the kinetics of tyrosine phos- similar NFAT activation in response to collagen (Fig. 3A, phorylation in response to collagen and convulxin are not due upper panel). In contrast, convulxin induced NFAT through to differential activation-induced cleavage of GPVI from the GPVI–FcRc only (Fig. 3A, lower panel), indicating that cell surface, which has been reported in platelets [28–30], convulxin is not a ligand for LAIR-1. The elevated NFAT because GPVI expression was not reduced for up to 60 min activity in unstimulated cells that expressed GPVI–FcRc and poststimulation (Fig. 2D, lower panels). Together, these data LAIR-1–CD3f (Fig. 3A, lower panel; note the difference in y- demonstrate that DT40 is a suitable model cell line in which to axis scales from the upper panel) is consistent with previously study GPVI signaling in response to collagen. reported basal signaling by ITAM receptors [34]. To then determine whether LAIR-1 specifically inhibits GPVI collagen signaling, GPVI–FcRc NFAT assays were carried out in DT40 The inhibitory collagen receptor LAIR-1 inhibits GPVI cells in the presence or absence of LAIR-1 transfection. signaling in response to collagen but not convulxin Strikingly, LAIR-1 inhibited the GPVI response to collagen by A potential explanation for the relatively strong collagen over 90% (Fig. 3B, upper panel) but had no effect on signaling in DT40 B-cells vs. RBL-2H3 mast cells is the convulxin signaling (Fig. 3B, lower panel). Similar to collagen,

AB40 30 Unstimulated 30 Unstimulated Collagen Collagen 20 20 10 10 0 0 0.8 0.6 Unstimulated 0.6 Unstimulated 0.4 Convulxin Convulxin 0.4 NFAT-Iuciferase activity NFAT-Iuciferase activity (percent PMA + ionomycin)

0.2 (percent PMA + ionomycin) 0.2 0 0 γ γ γ ζ LAIR-1 Control Control + LAIR-1 GPVI/FcR GPVI/FcR GPVI/FcR LAIR-1-CD3

Fig. 3. The inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) inhibits glycoprotein (GP)VI–FcRc responses to collagen but not convulxin. (A) The DT40 B-cell line was transfected with a nuclear factor of activated T-cells (NFAT)–luciferase reporter construct, a b-galactosidase construct to control for transfection efficiency, and 2 lg each of expression constructs for GPVI and FcRc, or leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1)–CD3f, or empty vector controls. Cells were stimulated with 10 lgmL)1 collagen (upper panel) or 10 lgmL)1 convulxin (lower panel), and NFAT–luciferase assays were performed as described in the legend to Fig. 1C. The luciferase data are presented as a percentage of the positive control PMA plus ionomycin response. These PMA plus ionomycin controls and b-galactosidase levels were similar for each transfection, suggesting similar transfection efficiencies (data not shown). Error bars represent the standard error of the mean from three independent experiments. (B) NFAT–luciferase reporter assays were performed on GPVI–FcRc-transfected DT40 cells as described above, but in the presence or absence of LAIR-1 transfection. LAIR-1 expression did not affect GPVI expression levels, as detected by flow cytometry (data not shown). Error bars represent the standard error of the mean from three independent experiments.

2007 International Society on Thrombosis and Haemostasis 2280 M. G. Tomlinson et al

CRP activated NFAT through LAIR-1–CD3f, consistent with A previous data [21], and CRP signaling through GPVI was Control inhibited by LAIR-1 (data not shown). Importantly, LAIR-1 expression did not alter GPVI levels as detected by flow cytometry (data not shown). Together, these data suggest that PP1 the absence of endogenous LAIR-1 in DT40 cells could explain 0 1.7 3.3 5.8 10 15 the stronger signaling response to collagen, relative to that in Time post treatment (min) the LAIR-1-expressing RBL-2H3 cells. B )

2 50 Collagen induces relatively weak but sustained 45 Control phosphotyrosine signaling in platelets 40 We have previously shown that the early tyrosine phosphory- 35 lation events induced by collagen in platelets are inhibited by 30 PP1 secondary mediator inhibition and a b and a b blockade, 2 1 IIb 3 25 whereas the convulxin response remains intact [6]. To determine

Platelet surface area (µm Platelet surface 20 whether collagen can promote a sustained signal in platelets, we 0 51015 measured GPVI-induced tyrosine phosphorylation over a 1-h Time post treatment (min) time course in the presence of inhibitors of the secondary Fig. 5. The maintenance of platelet spreading on collagen requires mediators, ADP and TXA2,anda2b1. EGTA was also included sustained signaling. (A) Human washed platelets, in the presence of to prevent platelet aggregation and subsequent aIIbb3 signaling, )1 2+ apyrase (2 U mL ), indomethacin (10 lM) and integrilin (9 lM), were and to inhibit Ca -dependent metalloproteinases that could allowedtospreadonacollagen-coated surface and observed in real time induce GPVI cleavage [29]. Similar to the cell line data (Figs 1B using Nomarski differential interference contrast microscopy. Platelets and 2D), collagen induced a weak, delayed but sustained were allowed to spread for 15 min, and maximally spread platelets were increase in tyrosine phosphorylation, whereas convulxin treated with dimethylsulfoxide vehicle control (upper panels) or 20 lM Src induced a relatively strong response that diminished over time family kinase inhibitor PP1 (lower panels) and followed for a further 15 min. Representative images of a single platelet are shown at the indi- (Fig. 4; major increases in tyrosine phosphorylation are indi- cated time points after treatment. The linear collagen fibers are also visible. cated by asterisks). We conclude from these data that collagen Similar data were obtained using 10 lM PP1 (data not shown). (B) induces relatively weak but sustained phosphotyrosine signaling Experiments were performed as described above for 11 control and 10 through GPVI in both cell lines and platelets. PP1-treated platelets, composed of three or four platelets from each of three donors. Images were captured every 50 s. The data show the mean platelet surface area over time post-treatment, and the error bars represent Sustained signaling maintains platelet spreading the standard error. Statistical analysis of the final 15-min time point demonstrated that PP1-treated platelets were significantly less spread than To further investigate the significance of the sustained signaling controls (P < 0.05; two-tailed t-test). by collagen, we tested the effect of interrupting sustained

Collagen Convulxin signaling on platelet spreading on collagen using the Src family 0 1' 10' 60' 0 1' 10' 60' kinase inhibitor PP1, which has been shown to inhibit GPVI 20'' 3' 30' 20'' 3' 30' signaling [35]. Platelets were allowed to spread on a collagen- 175 * coated surface in the presence of the aIIbb3 antagonist integrilin and inhibitors of the secondary mediators ADP and TXA2. 83 * Spreading was monitored using real-time video microscopy 62 and PP1, or its solvent was added after the platelets had 48 generated full lamellipodia. Strikingly, platelets retracted their * 33 lamellipodia upon PP1 addition, whereas control platelets 25 maintained their spread morphology as shown in Fig. 5A. This 17 7 effect was found to be statistically significant when mean surface areas were measured from multiple experiments Fig. 4. Collagen induces relatively delayed but sustained tyrosine phos- (Fig. 5B). These findings suggest that a sustained signal is phorylation in platelets. Human washed platelets, in the presence of 1 mM )1 )1 EGTA, 2 U mL apyrase, 10 lM indomethacin and 10 lgmL anti-a2 required to maintain platelet lamellipodia formation. blocking monoclonal antibody 6F1, were stimulated with collagen (30 lgmL)1) or convulxin (10 lgmL)1), and lyzed at the indicated time points, and proteins were separated by sodium dodecylsulfate polyacryl- Discussion amide gel electrophoresis and western blotted using the anti-phosphoty- rosine mAb 4G10. Asterisks indicate tyrosine phosphorylation that is The GPVI–FcRc complex is the main signaling receptor for sustained for collagen or transient for convulxin stimulation. The results collagen in platelets, promoting rapid and powerful platelet are representative of six experiments. activation at sites of injury. However, GPVI–FcRc is not a

2007 International Society on Thrombosis and Haemostasis Collagen promotes sustained GPVI signaling 2281 powerful signaling receptor in the absence of signals from on a single collagen-responsive RBL-2H3 clone [8], which feedback mediators. For example, collagen stimulates a small might have behaved atypically because of, for example, the increase in early tyrosine phosphorylation in platelets in the spontaneous loss of a negative regulator such as LAIR-1. An presence of inhibitors of the secondary feedback agonists, ADP alternative explanation is that, unlike NFAT activation, 2+ and TXA2, and blockade of the collagen-binding integrin a2b1, detectable Ca mobilization may truly require a threshold in contrast to the powerful response to the GPVI-reactive level of GPVI. Nevertheless, our cell line NFAT data are snake venom toxin convulxin [6]. Collagen is similarly unable consistent with two studies in platelets that reported collagen to induce a marked increase in Ca2+ mobilization in the signaling despite an 80% or 98% reduction in GPVI levels presence of secondary mediator inhibitors [7]. [9,10]. In this study, we have presented further evidence that The DT40 model B-cell line has allowed us to investigate the engagement of GPVI–FcRc by collagen induces weak signaling GPVI–FcRc signaling mechanism, as Kurosaki et al. have in platelets, by measuring whole cell protein tyrosine phos- previously generated lines deficient in key signaling molecules phorylation. We speculate that such weak signaling may be by homologous recombination [15–17]. Collagen-induced advantageous in reducing the risk of spontaneous thrombosis NFAT activation was entirely dependent on PLCc2andthe through non-specific crosslinking of the receptor. Indeed, the tyrosine kinase Syk, consistent with their absolute requirements platelet appears to have evolved to use a number of activating for collagen-induced platelet activation [27]. In the absence of receptors that on their own signal weakly, e.g. the von the tyrosine kinase Btk, NFAT activation was largely although Willebrand factor receptor GPIb-IX-V [36]. Importantly, these not entirely abrogated, again similar to platelet activation [27], receptors can act in concert with other glycoprotein and G- most likely because of compensation by another family protein-coupled receptors at sites of injury to promote positive member, Tec, which is expressed in both DT40 cells (M. G. feedback mediators and thus powerful platelet activation. After Tomlinson, unpubl. data) and platelets [27]. Also comparable activation by collagen, we have demonstrated that GPVI– to platelets, FcRc was required for GPVI expression on the cell FcRc induces sustained tyrosine kinase signaling and that surface (data not shown) [25]. Taken together, these data sustained Src kinase-based signaling is required for the suggest that collagen activates NFAT using a similar signaling maintenance of platelet lamellipodia. As GPVI–FcRc is the pathway as used for platelet activation, which leads us to main signaling receptor for collagen on platelets, we propose propose that NFAT activation in cell lines is a good surrogate that sustained signaling through this receptor plays an impor- for GPVI–FcRc signaling in platelets. tant role in maintenance of lamellipodia, but we cannot rule Our comparisons of collagen and convulxin signaling in out the possibility that a2b1 Ôoutside-inÕ signaling [37] is also platelets and cell lines showed that whereas collagen induced involved. relatively weak tyrosine phosphorylation that was delayed but The finding that collagen promotes weak but sustained sustained, possibly because of a failure to reach a threshold tyrosine phosphorylation through GPVI–FcRc in platelets has required to activate negative feedback pathways, convulxin enabled us to develop a cell line model system to monitor GPVI induced strong, rapid and transient phosphorylation. Mecha- signaling. We used an NFAT transcriptional reporter, which is nistically, this may be due to a relatively weak collagen–GPVI widely used to measure ITAM signaling responses over a interaction that requires the avidity effect of GPVI dimeriza- period of several hours and is effectively activated by a weak tion [38–40]. Moreover, the minimal glycine–proline–hydroxy- signal that is sustained [13]. Indeed, we found that collagen proline (GPO) motifs that mediate firm GPVI binding of induced NFAT activation through GPVI–FcRc in RBL-2H3 sequence GPOGPO [41] are not abundant along the length of cells, despite an absence of detectable Ca2+ mobilization. The each triple helical collagen fiber, suggesting relatively weak time course of tyrosine phosphorylation in this cell line further GPVI crosslinking. In contrast, convulxin is multimeric, supports the model in which GPVI–FcRc promotes weak but existing as a tetramer of heterodimers [42,43], suggesting that sustained signaling in response to collagen. it can cause extensive clustering of GPVI to promote rapid and Before the present study, several groups had attempted to strong activation. Paradoxically, the strongest NFAT activa- measure collagen-induced GPVI signaling in cell lines by tion was observed in GPVI–FcRc-transfected DT40 cells measuring Ca2+ mobilization and tyrosine phosphorylation stimulated with collagen. We propose that this is due to a over short time courses. Although we and others failed to combination of the sustained signaling by collagen that is observe any Ca2+ signaling [4,5], two groups were successful, optimal for NFAT activation [13] and the absence of endog- although the collagen responses were weak in comparison to enous LAIR-1 expression on DT40 B-cells. LAIR-1 is a responses to the GPVI-activating snake venom toxin convulxin recently identified collagen receptor [21] that can negatively [2,8]. One of these reports proposed that a threshold density of regulate ITAM signaling through two cytoplasmic ITIM GPVI, approximating to that on platelets, was required to motifs that recruit the tyrosine phosphatases SHP-1 and confer collagen responsiveness, as measured by Ca2+ mobili- SHP-2 [31] and the tyrosine kinase Csk [32]. This inhibitory zation [8]. Using the NFAT reporter assay system, however, we collagen receptor is expressed on most hematopoietic cell lines, found no evidence that a threshold level of GPVI was required including RBL-2H3 cells, but is absent from most B-cell lines for collagen signaling. A potential reason for the discrepancy and platelets [31–33]. We cannot completely rule out the between this and the previous study is that the latter was based existence of LAIR-1 on the chicken DT40 B-cell line, because

2007 International Society on Thrombosis and Haemostasis 2282 M. G. Tomlinson et al of the lack of an antibody to the chicken homolog, but our studies of human and mouse glycoprotein VI: a platelet-specific failure to find chicken LAIR-1 in current sequence databases collagen receptor from the immunoglobulin superfamily. Blood 2000; (data not shown) suggests that DT40 cells do not express this 96: 1798–807. 4 Zheng YM, Liu C, Chen H, Locke D, Ryan JC, Kahn ML. Expression molecule. Nevertheless, our finding that LAIR-1 is a potent of the platelet receptor GPVI confers signaling via the Fc receptor and specific inhibitor of GPVI collagen signaling, when gamma-chain in response to the snake venom convulxin but not to ectopically expressed in DT40 cells, suggests that an absence collagen. JBiolChem2001; 276: 12999–3006. of LAIR-1 could be responsible for the relatively strong 5 Berlanga O, Tulasne D, Bori T, Snell DC, Miura Y, Jung S, Moroi M, collagen signaling. Conversely, the inhibitory effect of endog- Frampton J, Watson SP. The Fc receptor gamma-chain is necessary andsufficienttoinitiatesignallingthroughglycoproteinVIintrans- enous LAIR-1 on RBL-2H3 mast cells may be responsible for fected cells by the snake C-type lectin, convulxin. Eur J Biochem 2002; the relatively weak or undetectable GPVI collagen responses 269: 2951–60. that we and others have observed in these and other cell lines 6 Atkinson BT, Jarvis GE, Watson SP. Activation of GPVI by collagen [4,5,8]. is regulated by alpha2beta1 and secondary mediators. JThromb In summary, we have used platelets and two hematopoietic Haemost 2003; 1: 1278–87. 7 Ohlmann P, Eckly A, Mangin P, Lanza F, Gachet C. Further evidence cell lines to show that collagen promotes sustained signaling that fibrillar collagen is unable to promote platelet shape change and through GPVI. Moreover, these findings have facilitated our aggregation in the absence of secondary agonists. J Thromb Haemost generation of the first robust model cell line system to study 2005; 3: 2119–21. GPVI signaling in response to collagen. The NFAT reporter 8 Chen H, Locke D, Liu Y, Liu C, Kahn ML. The platelet receptor GPVI assay that we have characterized is highly sensitive, is mediates both adhesion and signaling responses to collagen in a recep- tor density-dependent fashion. JBiolChem2002; 277: 3011–9. quantitative, and requires only transient transfection, and 9 Snell DC, Schulte V, Jarvis GE, Arase K, Sakurai D, Saito T, Watson therefore can in future be used to address structure–function SP, Nieswandt B. Differential effects of reduced glycoprotein VI levels relationships, including the functional consequences of GPVI on activation of murine platelets by glycoprotein VI ligands. Biochem J polymorphisms that predispose to thrombotic disease [44], and 2002; 368: 293–300. whether the recently proposed GPVI dimer formation [38,45] is 10 Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. MolCellBiol2003; 23: necessary for function. Additionally, and perhaps most 4764–77. importantly, this cell line model can be used for high- 11 Shapiro VS, Mollenauer MN, Greene WC, Weiss A. c-rel regulation of throughput screening of putative GPVI antagonists for the IL-2 gene expression may be mediated through activation of AP-1. J treatment of heart attack and stroke. Exp Med 1996; 184: 1663–9. 12 Macian F. NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 2005; 5: 472–84. Acknowledgements 13 Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential acti- vation of transcription factors induced by Ca2+ response amplitude We are grateful to members of the Watson Lab for their and duration. Nature 1997; 386: 855–8. advice and comments. We thank B. Coller for the anti-a2 14 Bori-Sanz T, Inoue KS, Berndt MC, Watson SP, Tulasne D. antibody, T. Kurosaki for the DT40 cell lines, M. Moroi for Delineation of the region in the glycoprotein VI tail required for association with the Fc receptor gamma-chain. JBiolChem2003; 278: the anti-GPVI mAb, D. Powner for the RBL-2H3 cell line, 35914–22. Y. Senis for crosslinking CRP, A. Weiss for the NFAT– 15 Takata M, Sabe H, Hata A, Inazu T, Homma Y, Nukada T, luciferase reporter construct, and V. Heath for critically Yamamura H, Kurosaki T. Tyrosine kinases Lyn and Syk regulate B 2+ reading the manuscript. cell receptor-coupled Ca mobilization through distinct pathways. EMBO J 1994; 13: 1341–9. 16 Takata M, Kurosaki T. A role for BrutonÕs tyrosine kinase in B cell Disclosure of Conflict of Interests antigen receptor-mediated activation of phospholipase C-gamma 2. JExpMed1996; 184: 31–40. M. G. Tomlinson is supported by an MRC New Investigator 17 Takata M, Homma Y, Kurosaki T. Requirement of phospholipase C- Award, S. D. Calaminus holds a BHF Studentship, and S. P. gamma2activationinsurfaceimmunoglobulin M-induced B cell Watson holds a BHF Chair. O. Berlanga, J. M. Auger, T. Bori- . JExpMed1995; 182: 907–14. 18 McCarty OJ, Calaminus SD, Berndt MC, Machesky LM, Watson SP. Sanz and L. Meyaard state that they have no conflict of mediates platelet spreading through glycopro- interest. tein Ib and alpha(IIb)beta3 in the presence of botrocetin and ristocetin, respectively. JThrombHaemost2006; 4: 1367–78. 19 Moroi M, Mizuguchi J, Kawashima S, Nagamatsu M, Miura Y, References Nakagaki T, Ito K, Jung SM. A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous 1 Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the collagen. Thromb Haemost 2003; 89: 996–1003. central receptor? Blood 2003; 102: 449–61. 20 Morton LF, Hargreaves PG, Farndale RW, Young RD, Barnes MJ. 2 Clemetson JM, Polgar J, Magnenat E, Wells TN, Clemetson KJ. The Integrin alpha 2 beta 1-independent activation of platelets by simple platelet collagen receptor glycoprotein VI is a member of the immu- collagen-like peptides: collagen tertiary (triple-helical) and quaternary noglobulin superfamily closely related to FcalphaR and the natural (polymeric) structures are sufficient alone for alpha 2 beta 1-indepen- killer receptors. JBiolChem1999; 274: 29019–24. dent platelet reactivity. Biochem J 1995; 306: 337–44. 3 Jandrot-Perrus M, Busfield S, Lagrue AH, Xiong X, Debili N, Chic- 21 Lebbink RJ, de Ruiter T, Adelmeijer J, Brenkman AB, van Helvoort kering T, Le Couedic JP, Goodearl A, Dussault B, Fraser C, JM, Koch M, Farndale RW, Lisman T, Sonnenberg A, Lenting PJ, Vainchenker W, Villeval JL. Cloning, characterization, and functional

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Meyaard L. are functional, high affinity ligands for the 34 Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, inhibitory immune receptor LAIR-1. JExpMed2006; 203: 1419–25. Brown PO, Weiss A. T cell receptor-independent basal signaling via 22 Verbrugge A, Ruiter Td T, Clevers H, Meyaard L. Differential Erk and Abl kinases suppresses RAG gene expression. PLoS Biol contribution of the immunoreceptor tyrosine-based inhibitory 2003; 1:E53. motifs of human leukocyte-associated Ig-like receptor-1 to inhibi- 35 Quek LS, Pasquet JM, Hers I, Cornall R, Knight G, Barnes M, Hibbs tory function and phosphatase recruitment. Int Immunol 2003; 15: ML, Dunn AR, Lowell CA, Watson SP. Fyn and Lyn phosphorylate 1349–58. the Fc receptor gamma chain downstream of glycoprotein VI in 23 Tomlinson MG, Kane LP, Su J, Kadlecek TA, Mollenauer MN, Weiss murine platelets, and Lyn regulates a novel feedback pathway. Blood A. Expression and function of Tec, Itk, and Btk in lymphocytes: evi- 2000; 96: 4246–53. dence for a unique role for Tec. MolCellBiol2004; 24: 2455–66. 36 Ozaki Y, Asazuma N, Suzuki-Inoue K, Berndt MC. Platelet GPIb-IX- 24 McCarty OJ, Zhao Y, Andrew N, Machesky LM, Staunton D, V-dependent signaling. JThrombHaemost2005; 3: 1745–51. Frampton J, Watson SP. Evaluation of the role of platelet integrins in 37 Inoue O, Suzuki-Inoue K, Dean WL, Frampton J, Watson SP. Inte- fibronectin-dependent spreading and adhesion. JThrombHaemost grin alpha2beta1 mediates outside-in regulation of platelet spreading 2004; 2: 1823–33. on collagen through activation of Src kinases and PLCgamma2. JCell 25 Nieswandt B, Bergmeier W, Schulte V, Rackebrandt K, Gessner JE, Biol 2003; 160: 769–80. Zirngibl H. Expression and function of the mouse collagen receptor 38 Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson glycoprotein VI is strictly dependentonitsassociationwiththe MG, Watson SP. GPVI oligomerisation in cell lines and platelets. J FcRgamma chain. JBiolChem2000; 275: 23998–4002. Thromb Haemost 2007; 5: 1026–33. 26 Kurosaki T. Genetic analysis of B cell antigen receptor signaling. Annu 39 Miura Y, Takahashi T, Jung SM, Moroi M. Analysis of Rev Immunol 1999; 17: 555–92. the interaction of platelet collagen receptor glycoprotein VI (GPVI) 27 Watson SP, Auger JM, McCarty OJ, Pearce AC. GPVI and integrin with collagen. A dimeric form of GPVI, but not the monomeric alphaIIb beta3 signaling in platelets. JThrombHaemost2005; 3: 1752– form, shows affinity to fibrous collagen. JBiolChem2002; 277: 46197– 62. 204. 28 Bergmeier W, Rabie T, Strehl A, Piffath CL, Prostredna M, Wagner 40 OÕConnor MN, Smethurst PA, Davies LW, Joutsi-Korhonen L, Onley DD, Nieswandt B. GPVI down-regulation in murine platelets through DJ, Herr AB, Farndale RW, Ouwehand WH. Selective blockade of metalloproteinase-dependent shedding. Thromb Haemost 2004; 91: glycoprotein VI clustering on collagen helices. JBiolChem2006; 281: 951–8. 33505–10. 29 Gardiner EE, Arthur JF, Kahn ML, Berndt MC, Andrews RK. 41 Smethurst PA, Onley DJ, Jarvis GE, OÕConnor MN, Knight CG, Herr Regulation of platelet membrane levels of glycoprotein VI by a AB,OuwehandWH,FarndaleRW.Theplatelet–collagen interaction: platelet-derived metalloproteinase. Blood 2004; 104: 3611–7. the smallest effective motif for glycoprotein VI-mediated platelet 30 Stephens G, Yan Y, Jandrot-Perrus M, Villeval JL, Clemetson KJ, adhesion and activation contains two glycine-proline-hydroxyproline Phillips DR. Platelet activation induces metalloproteinase-dependent triplets. JBiolChem2006; 282: 1296–304. GP VI cleavage to down-regulate platelet reactivity to collagen. Blood 42 Murakami MT, Zela SP, Gava LM, Michelan-Duarte S, Cintra AC, 2005; 105: 186–91. Arni RK. Crystal structure of the platelet activator convulxin, a 31 Meyaard L, Adema GJ, Chang C, Woollatt E, Sutherland GR, Lanier disulfide-linked alpha4beta4 cyclic tetramer from the venom of Cro- LL, Phillips JH. LAIR-1, a novel inhibitory receptor expressed on talus durissus terrificus. Biochem Biophys Res Commun 2003; 310: 478– human mononuclear leukocytes. Immunity 1997; 7: 283–90. 82. 32 Verbrugge A, Rijkers ES, de Ruiter T, Meyaard L. Leukocyte- 43 Batuwangala T, Leduc M, Gibbins JM, Bon C, Jones EY. Structure of associated Ig-like receptor-1 has SH2 domain-containing phosphatase- the snake-venom toxin convulxin. Acta Crystallogr D Biol Crystallogr independent function and recruits C-terminal Src kinase. Eur J 2004; 60: 46–53. Immunol 2006; 36: 190–8. 44 Yee DL, Bray PF. Clinical and functional consequences of platelet 33 van der Vuurst de Vries AR, Clevers H, Logtenberg T, Meyaard L. membrane glycoprotein polymorphisms. SeminThrombHemost2004; Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) 30: 591–600. is differentially expressed during human B cell differentiation and 45 Horii K, Kahn ML, Herr AB. Structural basis for platelet collagen inhibits B cell receptor-mediated signaling. Eur J Immunol 1999; 29: responses by the immune-type receptor glycoprotein VI. Blood 2006; 3160–7. 108: 936–42.

2007 International Society on Thrombosis and Haemostasis