Journal of Thrombosis and Haemostasis, 5: 1026–1033

ORIGINAL ARTICLE Glycoprotein VI oligomerization in cell lines and

O. BERLANGA,1 *T.BORI-SANZ,1 *J.R.JAMES, J. FRAMPTON,* S. J. DAVIS, M. G. TOMLINSON* and S . P . W A T S O N * *Institute of Biomedical Research, Medical School, University of Birmingham, Edgbaston, Birmingham; and Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, University of Oxford, Oxford, UK

To cite this article: Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson MG, Watson SP. Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost 2007; 5: 1026–33. mediated through two distinct classes on the

Summary. Background: Glycoprotein VI (GPVI) is a physio- surface, the integrin aIIb1 and the glycoprotein VI (GPVI)–FC logic receptor for expressed at the surface of platelets receptor (FcR) c-chain receptor complex [1]. Blocking of either and megakaryocytes. Constitutive dimerization of GPVI has receptor in vitro using specific antibodies inhibits or delays, been proposed as being necessary for the interaction with respectively, collagen-induced platelet aggregation [2–4]. Simi- collagen, although direct evidence of dimerization has not been larly, platelets deficient in aIIb1 or GPVI–FcR c-chain show reported in cell lines or platelets. Objectives: To investigate loss of reactivity towards collagen in vitro [5–8]. Furthermore, oligomerization of GPVI in transfected cell lines and in platelets mice deficient in GPVI–FcR c-chain are protected against under non-stimulated conditions. Methods and results: By lethal thromboembolism [8,9], illustrating the crucial role that using a combination of molecular and biochemical techniques, the receptor plays in vivo under pathologic conditions. we demonstrate that GPVI association occurs at the surface of Although the intracellular signaling events mediated by aIIb1 transfected 293T cells under basal conditions, through an in platelets have remained elusive [10,11], the mechanism of interaction at the extracellular domain of the receptor. action of GPVI has been well documented and remains an area Bioluminescence resonance energy transfer was used to confirm of intense research. GPVI mediates platelet activation in oligomerization of GPVI under these conditions. A chemical response to collagen through a pathway that shares many crosslinker was used to detect constitutive oligomeric forms of features with those used by immune receptors such as FceRI, GPVI at the surface of platelets, which contain the Fc receptor and T-cell and B-cell antigen receptors [12]. As GPVI has no (FcR) c-chain. Conclusions: The present results directly dem- intrinsic signaling capacity, it is widely recognized that it must be onstrate GPVI–FcR c-chain oligomerization at the surface of coexpressed in association with the FcR c-chain, which acts as the platelet, and thereby add to the growing evidence that the signaling partner. Furthermore, this association is a prere- oligomerization of GPVI may be a prerequisite for binding of quisite for surface expression of GPVI on mouse platelets [13]. the receptor to collagen, and therefore for proper functioning of Froma structure–function pointofview,thereare several lines platelets upon vascular damage. of circumstantial evidence to suggest that GPVI functions as a dimer on the platelet surface. Moroi and coworkers have Keywords: BRET, dimerization, glycoprotein VI, GPVI, olig- demonstrated, using recombinant protein, that collagen binds omerization, platelets. to the dimeric but not the monomeric form of GPVI, and that only the former is able to attenuate collagen-induced platelet Introduction aggregation [14]. In contrast, both the monomeric and dimeric formsofGPVIbindtoimmobilizedconvulxinandinhibitplatelet The protein collagen is the major and most aggregation induced by the snake toxin with similar concentra- thrombogenic component of the vessel wall. Circulating tion dependencies [14]. The possibility that GPVI functions as a platelets adhere to exposed collagen and undergo activation, dimer is strongly reinforced by studies analyzing the ability of a leading to thrombus formation. The interaction with collagen is series of synthetic peptides with differentially spaced GPVI- recognition motifs to activate the collagen receptor in platelets Correspondence: Steve P. Watson, Institute of Biomedical Research, [15]. Finally, structural studies of the two immunoglobulin (Ig) Medical School, University of Birmingham, Edgbaston, Birmingham domains of human GPVI have revealed the formation of a back- B15 2TT, UK. to-back dimer in the crystal structure, which is mediated through Tel.: +44 0 121 414 6514; fax: +44 0 121 415 8817; e-mail: s.p. the more membrane-proximal of the two Ig domains [16]. [email protected] As the FcR c-chain is present as a disulfide-linked homo- 1These authors contributed equally to this work. dimer, it has been proposed that each chain associates independently with GPVI [17]. In light of a recent report Received 13 May 2006, accepted 9 February 2007 indicating that the two chains of the FcR c-chain are necessary

2007 International Society on Thrombosis and Haemostasis Platelet glycoprotein VI oligomerization 1027 for binding a single GPVI molecule [18], this model needs to be Approximately 10 days later, individual clones of cells were reviewed. Furthermore, direct evidence that GPVI is expressed selected and placed in 96-well plates for expansion. at the cell surface as a dimer or possibly as a larger complex is not available. We set out to investigate this in platelets and in Plasmid constructs transfected cell lines using distinct biochemical and molecular approaches. Our results confirm that GPVI is capable of GPVI–Flag and (D288)GPVI–Flag were subcloned into the pRc undergoing oligomerization in transfected cells and forming plasmid, whereas the FcR c-chain was subcloned into the pMG oligomers in platelets, and indicate that a modified version of plasmid, as previously described [19]. GPVI–Myc was obtained the current model for GPVI dimerization may be necessary. by standard PCR using a vector primer (T7) and GPVI–Myc primer (5¢-CCCTAAGCGGCCGCTCACAGATCCTCTT- CTGAGATGAGTTTTTGTTCTGAACATAACCCGCGG- Materials and methods C-3¢). The final amplified product was digested with HindIII and NotI, and inserted into the similarly cut mammalian expression Reagents and antibodies vector pcDNA 3.1. The CD2 extracellular domain was fused to Convulxin was purchased from Latoxan (Valence, France). the transmembrane region and cytoplasmic tail of GPVI using Anticonvulxin was a kind gift from M. Leduc (Institute standard overlapping PCR techniques. The extracellular Pasteur, Paris, France). Anti-CD2 antibody was kindly domain of CD2 was amplified using oligo 1 (5¢-CCCTAA- supplied by V. Horejsi (Institute of Molecular Genetics, AAGCTTACCATGAGCTTTCCATGTAAATTT-3¢)and Academy of Sciences, Prague, Czech Republic). Anti-FcR c- oligo 2 (5¢-GGTGTAGTAGTCCAGACCTTTCTCTGGA- chain was obtained from Upstate Biotechnology (Buckingham, CA-3¢), and a fragment containing the transmembrane and UK). Anti-Flag (M2) was obtained from Sigma (Dorset, UK). intracellular domains of GPVI was amplified using oligo 3 (5¢- Anti-Myc (9B11) was obtained from Cell Signalling Technol- GGTCTGGACTACTACACCAAGGGCAACCTG-3¢)and ogy (Hertfordshire, UK). All other reagents were obtained a vector primer (sp6). The two fragments were subsequently from previously described sources [19] unless otherwise stated. mixed together, and oligo 1 and sp6 were added to perform a second overlap PCR. The final amplified product, encoding a chimeric protein containing the extracellular part of CD2 and Platelet preparation the transmembrane and intracellular domains of GPVI, was Human blood was taken from drug-free volunteers on the day digested with HindIII and XbaI and inserted into the similarly of experiment using acidic citrate dextrose (120 mM sodium cut mammalian expression vector pcDNA 3.1. GPVI–green citrate, 110 mM glucose, 80 mM citric acid). Platelet-rich fluorescent protein (GFP) and GPVI–luciferase constructs used plasma was obtained by centrifugation at 200 · g for 20 min, for bioluminescence resonance energy transfer (BRET) analysis and platelets were isolated by centrifugation at 1000 · g for were generated by excision of GPVI from pRc–GPVI–Flag, ) 10 min in the presence of prostacyclin (0.1 lgmL 1). Platelets followed by cloning into pGFP2-N3 and pRluc-N3 (PerkinEl- were resuspended in modified TyrodeÕs/HEPES buffer mer, UK), respectively. CD2 and cytotoxic T-lymphocyte- (134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4.12H2O, associated antigen-4 (CTLA-4) BRET constructs were prepared 12 mM NaHCO3,20mM HEPES, 1 mM MgCl2,5 mM glucose, as previously described [20]. The integrity and authenticity of ) pH 7.3, at 37 C) in the presence of prostacyclin (0.1 lgmL 1), constructs was confirmed by nucleotide sequencing. recentrifuged at 1000 · g for 10 min, and resuspended in the ) above buffer to a density of 5 · 108 cells mL 1. Immunoprecipitation Stimulations were terminated by the addition of an equal Cell culture volume of ice-cold lysis buffer (2% Nonidet P-40, 300 mM

293T cells were grown in DMEM supplemented with NaCl, 20 mM Tris, 10 mM EDTA, 2 mM Na3VO4,1mM ) ) 100 U mL 1 penicillin, 100 lgmL 1 streptomycin and 10% phenylmethanesulfonyl fluoride, 10 lgmL)1 leupeptin, )1 )1 heat-inactivated fetal bovine serum under 5% CO2/95% air in 10 lgmL aprotinin, and 1 lgmL pepstatin A, pH 7.4). a humidified incubator. Cells were kept at the exponential Insoluble cell debris was removed by centrifugation for 15 min phase of growth. at 12 000 · g at 4 C. Samples were immunoprecipitated using an appropriate antibody overnight, and 30 lL of 50% (v/v) protein A–sepharose or protein G–sepharose was then added Transfections for 2 h. Beads were washed with lysis buffer, and resuspended Cells were transfected using the calcium phosphate method as in 20 lL of sodium dodecylsulfate sample buffer. previously described [19], and incubated in complete medium for 48 h prior to experimentation. For stable transfections, Immunoblotting and blotting plasmid DNA was cut with an appropriate restriction enzyme, and after transfection following the above method, divided into Samples were separated by sodium dodecylsulfate polyacryl- 24 wells; the required antibiotic for selection was then added. amide gel electrophoresis (SDS–PAGE) and transferred to

2007 International Society on Thrombosis and Haemostasis 1028 O. Berlanga et al poly(vinylidene difluoride) membranes, and then blocked with The reaction was quenched by adding Tris–HCl (pH 7.5) at a TBS-T [0.5 M Tris, 1.5 M NaCl, 0.1% (v/v) Tween-20, pH 7.4] final concentration of 50 mM for 20 min. Samples were lyzed containing 10% (w/v) bovine serum albumin (BSA) for at least and separated by SDS–PAGE under non-reducing conditions. 1 h. Blots were incubated with appropriate antibodies and developed using an enhanced chemiluminescence (ECL) detec- BRET tion system. For GPVI ligand blotting with convulxin, membranes were incubated with 10 lgmL)1 of convulxin for The BRET analysis was carried out essentially as previously 1 h at room temperature, and incubated with anticonvulxin described [20]. Briefly, FuGene (Roche, Hertfordshire, UK) was antibody. Prestained molecular weight protein markers were used to transfect 293T cells, with varying ratios of the GFP and obtained from Bio-Rad (Hemel Hempstead, UK) and New luciferase constructs. Cells were harvested 24 h post-transfec- England Biolabs (Hertfordshire, UK). tion, and, for each transfection, 10 lM DeepBlueC (final concentration) was added to 100 lL of cells in a 96-well plate, and light emission in the 410 ± 40-nm (LU-A) and 515 ± 15- Flow cytometry nm (LU-B) wavelength ranges was measured immediately. GFP Cells were resuspended in phosphate-buffered saline (PBS) and luciferase expression was measured in a separate well, and )1 containing 1 mg mL BSA. Cells were incubated with converted to a ratio of concentrations. BRETeff values were 10 lgmL)1 of primary antibody for 15 min, washed, and calculated, after background subtraction, as LU-B/LU-A, incubated with fluorescein isothiocyanate-conjugated secon- corrected for luciferase expression alone (typically 7% of LU- dary antibody for a further 15 min. Stained cells were analyzed A). To assess the ability of ligands to alter GPVI oligomeriza- immediately using FACScalibur (Becton Dickinson, Oxford, tion, 10 lL of convulxin (10 lgmL)1), collagen (20 lgmL)1) UK). Data were recorded and analyzed using CELLQUEST or PBS (control) was incubated with GPVI-transfected cells for software. 15 min before assaying for BRET as above.

Chemical crosslinking assay Results The crosslinking reaction was performed on washed platelets GPVI undergoes oligomerization in 293T cells resuspended in PBS at room temperature. Sulfo-EGS is a water- soluble analog of a homobifunctional N-hydroxysuccimide We sought to investigate the possible dimerization or forma- ester (NHS-ester), EGS. Primary amines are principal targets for tion of higher oligomers of GPVI in a transfected cell line via NHS-esters. Platelets were incubated with freshly prepared immunoprecipitation. Plasmid constructs were generated crosslinker solution at concentrations of 1 mM,1.5mM and coding for GPVI coupled to either Myc or Flag tags at the 2mM sulfo-EGS in PBS for 30 or 60 min at room temperature. cytosolic C-terminus (Fig. 1A), and transiently transfected into

A C

Mock GPVI-FlagGPVI-MycGPVI-Flag+GPVI-Myc IP: Flag WB: Myc 50

LB: GPVI 50

γ-chainγ-chain in +FcR ha GPVI-Flag GPVI-Myc γ-c γ-chain BD+FcR Mock GPVI-Flag GPVI-Myc FcR GPVI-Flag+FcRGPVI-MycGPVI-Flag+GPVI-Myc IP: Flag WB: Myc 50 IP: Flag WB: FcR γ-chain 20

GPVI LB: GPVI 50

FSC

Fig. 1. Glycoprotein VI (GPVI) undergoes oligomerization in 293T cells. (A) Schematic representation of GPVI–Flag and GPVI–Myc constructs. (B) Mock-transfected, GPVI–Flag-transfected or GPVI–Myc-transfected 293T cells were incubated with monoclonal antibody 204-11 and fluorescein isothiocyanate-labeled antimouse IgG. The fluorescence was analyzed by flow cytometry. Dot plots were constructed for forward-scatter on the x-axis vs. fluorescence intensity, which is a marker for GPVI, on the y-axis. (C) 293T cells were transiently transfected with either GPVI–Flag, GPVI–Myc or both, and lyzed; protein lysates were then immunoprecipitated with anti-Flag antibody. Samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis under non-reducing conditions. Membranes were blotted with anti-Myc antibody in order to detect coprecipitated GPVI–Myc. Levels of GPVI-transfected protein were measured by convulxin ligand blot using whole cell lysates. (D) The same experiment was repeated but cotransfecting Fc receptor (FcR) c-chain. Membranes were blotted with anti-Myc antibody (top panel) and anti-FcR c-chain antibody (middle panel) in order to detect coprecipitated proteins. Levels of GPVI-transfected protein were measured by convulxin ligand blotting using whole cell lysates (lower panel). Results are representative of five experiments.

2007 International Society on Thrombosis and Haemostasis Platelet glycoprotein VI oligomerization 1029

293T cells, a human kidney cell line with a high efficiency of A CD2-GPVI-Flag (Δ288)GPVI-Flag transfection. The expression of both forms of GPVI was detected by western blotting for Myc and Flag (not shown). The two forms of GPVI were expressed at similar levels at the surface of the cells in the absence of FcR c-chain, as CD2 GPVI demonstrated by flow cytometry using an anti-GPVI antibody (Fig. 1B). In order to demonstrate association, 293T cells were transiently cotransfected with both GPVI constructs, either GPVI with or without FcR c-chain. After 2 days in culture, the cells were lyzed, and protein extracts were immunoprecipitated Flag using an anti-Flag antibody. Subsequent western blot analysis Flag using an anti-Myc antibody demonstrated association between the two tagged forms of GPVI (Fig. 1C), independent of FcR c-chain. Cotransfection of the latter did not increase the yield Mock cells CD2-GPVI-Flag of association (Fig. 1D). Significantly, the experiments shown B in Fig. 1C,D were performed on the same day, and the ECL exposures are time-matched. Ligand blots using convulxin revealed similar levels of expression of GPVI in the cotrans- fected cells. In order to rule out the possibility that association was an artefact of this experimental approach, lysates from Mock cells (Δ288)GPVI-Flag cells with either GPVI–Flag or GPVI–Myc were combined and subjected to immunoprecipitation and western blotting as above. Under these conditions, no association was detected (data not shown). These observations confirm that GPVI is GPVI able to undergo oligomerization in transfected cells, and that this is not dependent on the FcR c-chain. FSC

C Oligomerization takes place through the extracellular domain GPVI-Myc

GPVI-Myc of GPVI + GPVI-Flag+

Δ288) Further studies were performed to establish whether the CD2-GPVI-Flag+GPVI-Myc( GPVI-Flag intracellular or extracellular regions of GPVI are required for IP: Flag WB: Myc the homophilic interaction. For these studies, a chimeric and 50 a mutant protein were generated (Fig. 2A). The chimeric LB: GPVI protein consisted of the extracellular domain of the Ig surface 50 protein CD2 coupled to the transmembrane and intracellular WB: CD2 37 domains of GPVI, and tagged with Flag (CD2–GPVI–Flag) (Fig. 2A). For the mutant protein, GPVI was truncated at Fig. 2. Glycoprotein VI (GPVI) undergoes oligomerization through its the interface of the transmembrane and cytoplasmic domains, extracellular domain. (A) Diagram of CD2–GPVI–Flag (top-left) and and tagged with Flag at the C-terminus (D288)GPVI–Flag) (D288)GPVI–Flag (top-right). (B) The presence of either protein at the (Fig. 2A). Similar levels of expression of the two constructs surface of transfected cells was detected by flow cytometry using specific was demonstrated by transient transfection into 293T cells antibodies for CD2 or GPVI. (C) 293T cells were cotransfected with the indicated constructs. Protein lysates were immunoprecipitated using an followed by western or ligand blotting (not shown), and by anti-Flag antibody. Cotransfected full-length GPVI–Myc was detected by flow cytometry using specific antibodies for CD2 or GPVI western blotting using an anti-Myc antibody. Whole cell lysates were used (Fig. 2B). to assess levels of transfected GPVI (middle panel) and to detect expression The two modified forms of GPVI were cotransfected with of CD2–GPVI–Flag using an anti-CD2 antibody (lower panel), which Myc-tagged wild-type GPVI (GPVI–Myc) in 293T cells. runs at a lower molecular weight than the constructs that contain the GPVI extracellular domain. Results are representative of three experi- Protein association was detected by immunoprecipitation with ments. an antibody to Flag and western blotting for Myc (Fig. 2C). (D288)GPVI–Flag was found to coprecipitate with wild-type GPVI–Myc, demonstrating that the GPVI cytosolic tail is not GPVI oligomerization by BRET analysis required for the GPVI–GPVI interaction. In contrast, there was no association between CD2–GPVI and wild-type GPVI– BRET relies on the transfer of energy from a donor molecule Myc, confirming that the extracellular portion of GPVI is (luciferase) to an acceptor (GFP) that is only effective at essential for the homophilic interaction. distances of < 10 nm. Proteins of interest can be genetically

2007 International Society on Thrombosis and Haemostasis 1030 O. Berlanga et al

γ fused to these fluorophores, permitting the analysis of protein A GPVIBP +FcR -chain γ interactions in live cells, with the level of energy transfer 0.6 –FcR -chain

(BRETeff) and its dependence on the acceptor/donor ratio allowing the assignment of stoichiometry [20]. Human 293T cells were transiently cotransfected with GPVI–GFP 0.4

(GPVIGFP)-expressing and GPVI–luciferase (GPVILuc)-expres- eff sing plasmids as a ÔBRET pairÕ (GPVIBP), along with FcR c- BRET chain. Subsequent analysis demonstrated that GPVI was 0.2 capable of oligomerization, with the dependence of BRETeff values on the acceptor/donor ratio fitting best to a dimer model (Fig. 3A,B). The level of BRET observed was intermediate 0 between that of the known monomer, CD2, and the disulfide- 0 2 4 6 8 10 linked homodimer, CTLA-4, implying that GPVI dimers are [GFP]/[Luc] likely to be in equilibrium with the monomeric moiety at the CTLA-4 GPVI cell surface (Fig. 3A,B), as observed previously for CD80 [20]. B BP BP CD2 CD2 , GPVI 1.0 BP Luc GFP Expression of GPVI and CD2 as a BRET pair gave BRETeff values that were lower than those of CD2 and exhibited an independence from the acceptor/donor ratio, which is a 0.8 characteristic of random interactions at the cell surface [20].

eff 0.6 GPVI dimerization was observed regardless of FcR c-chain coexpression, although the level of dimerization was slightly

BRET 0.4 enhanced in its presence (Fig. 3A). Incubation of GPVI- transfected cells with either collagen or convulxin did not 0.2 increase the level of BRETeff observed for GPVI in the absence of ligand (Fig. 3C), with no effect being observed over 30 min 0 (not shown). This suggests that GPVI binding to these two 0 2 4 6 8 10 [GFP]/[Luc] ligands does not significantly alter the distance between luciferase- and GFP-coupled GPVI molecules measured in C GPVI BP + PBS BRET. 0.6 + Collagen + Convulxin

Chemical crosslinking of GPVI reveals the presence of high 0.4 molecular mass complexes in platelets eff To determine the molecular organization of GPVI on the BRET platelet surface, platelets were treated with a chemical cross- 0.2 linker that stabilizes pre-existing structures, including the presence of homodimers and associations with other mem- brane proteins. The membrane-impermeable crosslinker sulfo- 0 EGS was chosen for these studies, as it has been previously 0 2 4 6 8 10 [GFP]/[Luc] used in similar studies [21,22]. Three concentrations (1, 1.5 and Fig. 3. Bioluminescence resonance energy transfer (BRET) analysis of 2mM) of sulfo-EGS were assessed for incubation times of 30 293T cells transfected with glycoprotein VI (GPVI). (A) Cells were and 60 min as recommended by the manufacturer. Similar data cotransfected with GPVIGFP,GPVILuc and the Fc receptor (FcR) c-chain were obtained using all combinations, as illustrated following a (GPVIBP), to demonstrate the dependence of BRETeff values on the 30-minincubationwith1.5mM sulfo-EGS (Fig. 4), with no acceptor/donor ratio. These values were compared with the known major differences being seen between the 30-min and 60-min monomer, CD2, and the disulfide-linked homodimer, CTLA-4. Cotrans- time points. The subsequent separation of whole cell lysates by fection of GPVIGFP with CD2Luc showed an independence from acceptor/ donor ratio, as expected for random interactions. All data are shown fitted SDS–PAGE under non-reducing conditions, combined with to the most appropriate oligomerization model. BRETeff values from ligand blotting using the toxin, convulxin, GPVIBP - expressing cells in the presence or absence of FcRc-chain were identified three major molecular mass bands of approximately measured to assess the effect of the latter on GPVI dimerization. )1 )1 55, 70 and 220 kDa in sulfo-EGS-treated cells, in comparison (C) Incubation with collagen (20 lgmL ) or convulxin (10 lgmL )did to a single major band of around 55 kDa in control samples. In not increase the level of dimerization compared to a phosphate-buffered saline (PBS) control after 15 min. Results from three experiments are addition, several uncharacterized, minor bands could also be combined. seen that could be due to additional GPVI complexes, non- specific binding of convulxin, or the presence of contaminants blotting with an antibody to GPVI (not shown). The possibility in the snake toxin preparation. The major band of 55 kDa that the 70-kDa band corresponds to a complex of GPVI and a corresponds to the GPVI monomer, as confirmed by western dimer of FcR c-chain, which would be preserved under the

2007 International Society on Thrombosis and Haemostasis Platelet glycoprotein VI oligomerization 1031

A formation of a complex with a molecular mass greater than Blot: GPVI Sulfo-EGS – + 200 kDa, which appears to contain two prominent and other more minor bands. The size of this complex corresponds to 250 GPVI+FcR γ-chain (oligomer) that of the aIIbb3 heterodimer, with the multiple bands possibly 185 150 reflecting differential glycosylation. Significantly, however, as with GPVI and other platelet receptors [23], the monomeric 83 form of aIIb was the predominant band in the presence of the 75 GPVI+FcR γ-chain crosslinking reagent, which might reflect the inefficiency of the 62 GPVI (monomer) crosslinking process. 47

B Blot: FcR γ-chain Discussion Sulfo-EGS – + 250 The organization of the GPVI–FcR c-chain receptor complex 185 FcR γ-chain+GPVI (oligomer) at the surface of platelets is of considerable interest. GPVI has 75 been proposed as a possible target in the fight against 62 FcR γ-chain+GPVI 47 thrombus-related diseases. GPVI plays a recognized role in 32 platelet activation upon vascular injury under normal physi- 25 ologic conditions, and its absence has been linked to mild FcR γ-chain 17 bleeding disorders in humans and to protection against thrombus formation [24,25]. Furthermore, it has been pro- posed that GPVI antibodies and soluble GPVI dimers can C Blot: αIIb Sulfo-EGS – + protect against thrombus formation in animal models, although results for the latter have varied between different α IIb (heterodimer) research groups [26,27]. 175 It is widely accepted that GPVI must dimerize under basal αIIb (monomer) conditions in order to bind collagen, in view of the low affinity 83 of the monomeric form for the matrix protein. Furthermore, the concept of dimerization of GPVI is supported by a recent Fig. 4. Chemical crosslinking of platelets reveals the presence of oligomers report showing that the crystal structure of GPVI contains containing glycoprotein VI (GPVI). Platelets were incubated with sulfo- back-to-back dimers of the receptor through an interaction in EGS, and protein lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis under non-reducing conditions using the extracellular domain [16]. The accepted molecular model 8–20% (A, B) or 7.5% (C) acrylamide gels. (A) Ligand blot using for GPVI dimerization proposes that an FcR c-chain homodi- convulxin shows three major bands at around 55, 70 and 220 kDa. (B) mer binds a molecule of GPVI on either side [17], with the site Reblotting of the above membrane using an anti-Fc receptor (FcR) for interaction being in the transmembrane region, between the c-chain antibody demonstrates that the bands of 70 and 220 kDa contain aspartic acid of the FcR c-chain and the arginine residue of the FcR c-chain. These results indicate that the 55-kDa band is likely to represent a monomer of GPVI, the 70-kDa band will contain a monomer GPVI [19]. However, this model has recently been challenged of GPVI and a homodimer of FcR c-chain, and the 220-kDa band will by a study showing that the two aspartic residues in the FcR c- represent an oligomer containing both GPVI and FcR c-chain. (C) chain homodimer are necessary for association with the single Western blot from sulfo-EGS-treated cells using an anti-aIIb specific arginine residue in the transmembrane region of GPVI [18]. antibody reveals the presence of two major bands representing aIIb This model is energetically stable, as prior protonization of at monomer (lower band) and heterodimer (upper band). All blots were performed using two different molecular mass markers for better protein least one of the carboxyl groups in the aspartic acid may occur, size analysis. Results are representative of three experiments. so that there is no charge imbalance in the assembled structure [28,29]. Furthermore, this seems to be a distinctive but common non-reducing conditions of the experiment, was supported by assembly mechanism for a number of activating immune the appearance of a specific comigrating band in sulfo-EGS- receptors that are structurally similar to GPVI [18]. treated cells upon western blotting for FcR c-chain (Fig. 4B). Our results suggest that GPVI dimerizes at the surface of Similarly, the 220-kDa band was detected by western blotting GPVI-transfected 293T cells through an interaction that takes for FcR c-chain, suggesting that it may represent a trimeric place in the extracellular domain. FcR c-chain is not form of the GPVI–FcR c-chain dimer association [i.e. (GPVI– necessary for the dimerization, as it is not expressed in these

FcR c-chain)3]. Alternatively, it could represent the formation cells. This would agree with the proposed model for GPVI of a complex between GPVI–FcR c-chain and one or more dimerization from the recent crystallization study, which is other proteins on the platelet surface, or an alternative independent of FcR c-chain. Interestingly, in platelets, GPVI combination of GPVI and FcR c-chain. The efficiency of the requires association with FcR c-chain for expression on the crosslinker was investigated by heterodimerization of the platelet surface. However, this is not the case in the majority platelet integrin subunits aIIb and b3 (Fig. 4C). We show that of transfected cell lines that have been studied, as demon- under the same conditions, treatment with sulfo-EGS induces strated previously, not only for GPVI, but also for other

2007 International Society on Thrombosis and Haemostasis 1032 O. Berlanga et al immune receptors with similar structural organization that be a prerequisite for formation of a receptor complex that require FcR c-chain for expression in the host cell [19,30,31]. retains the capacity to bind collagen with high affinity and Although FcR c-chain is not essential for surface expression trigger a signal cascade inside the cell. Establishing the and dimerization of GPVI in the 293T cell line, it may help organization of this complex has important consequences for to stabilize expression, which may explain the difference in our understanding of platelet regulation by collagen and for dimerization in cells that express FcR c-chain, as demon- possible therapeutic strategies to fight cardiovascular disease. strated by BRET. Interestingly, experiments using platelets pretreated with a Acknowledgements chemical crosslinker that preserves surface complexes, followed by western blotting for either GPVI or FcR c-chain, demon- O. Berlanga was supported by the BHF. T. Bori-Sanz was strate the presence of two oligomeric structures that contain supported by the University of Birmingham (UK). J. R. James both proteins. The two bands of approximately 70 and and S. J. Davis are funded by the Wellcome Trust. M. G. 220 kDa containing GPVI and FcR c-chain could correspond Tomlinson is an MRC research fellow. S. P. Watson holds a to a single GPVI molecule with two FcR c-chains and a trimer BHF chair. of this complex, respectively. Alternatively, the latter could represent a protein multicomplex containing GPVI, FcR c- Disclosure of Conflict of Interests chain and possibly one or more other protein(s). The possibility that a GPVI trimer may be present in this oligomeric structure The authors states that they have no conflict of interest. is important, in that dimers show high affinity for fibrous collagen and inhibit platelet adhesion and aggregation to the References injured vessel wall in vivo [14,17,26]. An important question is the extent to which activation of 1 Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the GPVI by multivalent ligands is mediated by oligomerization. central receptor? Blood 2003; 102: 449–61. 2 Coller BS, Beer J, Scudder LE, Steinberg MH. Collagen–platelet The BRET results obtained indicate that neither convulxin interactions: evidence for a direct interaction of collagen with platelet nor collagen alter the average separation of GPVI molecules GPIa/IIa and an indirect interaction with platelet GPIIb/IIIa mediated at the cell surface. The recent crystal structures of convulxin by adhesive proteins. Blood 1989; 74: 182–92. and GPVI may provide an explanation for this [16,32]. The 3 Keely PJ, Parise LV. The alpha2beta1 integrin is a necessary co- structures indicate that only a monomer of convulxin can fit receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cgamma2 in platelets. JBiolChem into the binding site of GPVI. Furthermore, the tetrameric 1996; 271: 26668–76. shape of convulxin would not allow a GPVI dimer to form 4 Sugiyama T, Ishibashi T, Okuma M. Functional role of the antigen across the binding sites, because they are in the wrong recognized by an antiplatelet antibody specific for a putative collagen orientation, suggesting convulxin may bind monomeric GPVI. receptor in platelet–collagen interaction. Int J Hematol 1993; 58:99– On the other hand, the presence of multiple binding sites for 104. 5 Holtkotter O, Nieswandt B, Smyth N, Muller W, Hafner M, Schulte GPVI in convulxin would enable the snake toxin to crosslink V, Krieg T, Eckes B. Integrin alpha 2-deficient mice develop normally, GPVI molecules and thereby induce a signal into the cell. This are fertile, but display partially defective platelet interaction with col- underlines the fundamental difference between basal receptor lagen. JBiolChem2002; 277: 10789–94. dimerization/oligomerization, which appears to be required for 6 Moroi M, Jung SM, Okuma M, Shinmyozu K. A patient with platelets collagen binding, and agonist-induced receptor crosslinking/ deficient in glycoprotein VI that lack both collagen-induced aggrega- tion and adhesion. J Clin Invest 1989; 84: 1440–5. clustering, which is sufficient to induce signaling. 7 Nieuwenhuis HK, Akkerman JW, Houdijk WP, Sixma JJ . Human In the light of our own results as shown here, and those blood platelets showing no response to collagen fail to express surface recently published elsewhere [16,18], we propose a modified glycoprotein Ia. Nature 1985; 318: 470–2. version of the current model for GPVI dimerization at the 8 Kato K, Kanaji T, Russell S, Kunicki TJ, Furihata K, Kanaji S, platelet surface, whereby two GPVI molecules dimerize Marchese P, Reininger A, Ruggeri ZM, Ware J. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation through their respective membrane-proximal Ig domains in illustrated by targeted gene deletion. Blood 2003; 102: 1701–7. the extracellular domain, and each GPVI molecule binds an 9 Nieswandt B, Schulte V, Bergmeier W, Mokhtari-Nejad R, Racke- FcR c-chain homodimer in the transmembrane region. The brandt K, Cazenabe JP, Ohlmann P, Gachet C, Zimgibl H. Long-term two FcR c-chain homodimers must be kept apart under basal antithrombotic protection by in vivo depletion of platelet glycoprotein conditions, in order to avoid activation, but the latter can be VI in mice. JExpMed2001; 193: 459–69. 10 Inoue O, Suzuki-Inoue K, Dean WL, Frampton J, Watson SP. Inte- achieved upon collagen binding to initiate a signaling grin alpha2beta1 mediates outside-in regulation of platelet spreading response. on collagen through activation of Src kinases and PLCgamma2. JCell Overall, we have demonstrated that surface oligomerization Biol 2003; 160: 769–80. of GPVI in living cells occurs, and have provided evidence that 11 Bernardi B, Guidetti GF, Campus F, Crittenden JR, Graybiel AM, this may also take place in platelets, where a protein Balduini C, Torti M. The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3. multicomplex is formed that contains GPVI, FcR c-chain, Blood 2006; 107: 2728–35. and possibly one or more other component(s). The correct 12 Watson SP, Asazuma N, Atkinson B, Berlanga O, Best D, Bobe R, expression and assembly of this multimolecular structure may Jarvis G, Marshall S, Snell D, Stafford M, Tulasne D, Wilde J, Wonerow

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P, Frampton J. The role of ITAM- and ITIM-coupled receptors in 23 Newton JP, Hunter AP, Simmons DL, Buckley CD, Harvey DJ. platelet activation by collagen. Thromb Haemost 2001; 86: 276–88. CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated. 13 Nieswandt B, Bergmeier W, Schulte V, Rackebrandt K, Gessner JE, Biochem Bioph Res Com 1999; 261: 283–91. Zirngibl H. Expression and function of the mouse collagen receptor 24 Lockyer S, Okuyama K, Begum S, Le S, Sun B, Watanabe T, glycoprotein VI is strictly dependent on its association with the FcR- Matsumoto Y, Yoshitake M, Kambayashi J, Tandon NN. GPVI- gamma chain. JBiolChem2000; 275: 23998–4002. deficient mice lack collagen responses and are protected against 14 Miura Y, Takahashi T, Jung SM, Moroi M. Analysis of the interaction experimentally induced pulmonary thromboembolism. Throm Res of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A 2006; 118: 371–80. dimeric form of GPVI, but not the monomeric form, shows affinity to 25 Arai M, Yamamoto N, Moroi M, Akamatsu N, Fukutake K, Tanoue fibrous collagen. JBiolChem2002; 277: 46197–204. K. Platelets with 10% of the normal amount of glycoprotein VI have 15 Farndale RW, Siljander PR, Onley DJ, Sundaresan P, Knight CG, an impaired response to collagen that results in a mild bleeding ten- Barnes MJ. Collagen–platelet interactions: recognition and signalling. dency. Br J Haematol 1995; 89: 124–30. Biochem Soc Symp 2003; 70: 81–94. 26 Massberg S, Konrad I, Bu¨ ltmann A, Schulz C, Mu¨ nchG,PelusoM, 16 Horii K, Kahn ML, Herr AB. Structural basis for platelet collagen Lorenz M, Schneider S, Besta F, Mu¨ ller I, Hu B, Langer H, Kremmer responses by the immune-type receptor glycoprotein VI. Blood 2006; E, Rudelius M, Heinzmann U, Ungerer M, Gawaz M. Soluble 108: 936–42. glycoprotein VI dimer inhibits platelet adhesion and aggregation to the 17 Moroi M, Jung SM. Platelet glycoprotein VI: its structure and func- injured vessel wall in vivo. FASEB J 2004; 18: 397–9. tion. Throm Res 2004; 114: 221–33. 27 Gru¨ ner S, Prostredna M, Koch M, Miura Y, Schulte V, Jung SM, 18 Feng J, Garrity D, Call ME, Moffett H, Wucherpfennig KW. (2005) Moroi M, Nieswandt B. Relative antithrombotic effect of soluble Convergence on a distinctive assembly mechanism by unrelated fam- GPVI dimer compared with anti-GPVI antibodies in mice. Blood 2005; ilies of activating immune receptors. Immunity 2005; 22: 427–38. 105: 1492–9. 19 Berlanga O, Tulasne D, Bori T, Snell DC, Miura Y, Jung S, Moroi M, 28 Engelman DM. Electrostatic fasteners hold the T cell receptor–CD3 Frampton J, Watson SP. The Fc receptor gamma-chain is necessary complex together. Mol Cell 2003; 11: 5–6. and sufficient to initiate signalling through glycoprotein VI in trans- 29 Senes A, Engel DE, DeGrado WF. Folding of helical membrane fected cells by the snake C-type lectin, convulxin. Eur J Biochem 2002; proteins: the role of polar, GxxxG-like and proline motifs. Curr Opin 269: 2951–60. Struct Biol 2004; 14: 465–79. 20 James JR, Oliveira ML, Carmo AM, Iaboni A, Davis SJ. A rigorous 30 Taylor LS, McVicar DW. Functional association of FcepsilonRI- experimental framework for detecting protein oligomerization using gamma with arginine(632) of paired immunoglobulin-like receptor bioluminescence resonance energy transfer. Nat Methods 2006; 3: (PIR)-A3 in murine macrophages. Blood 1999; 94: 1790–6. 1001–6. 31 Ono M, Yuasa T, Ra C, Takai T. Stimulatory function of paired 21 Xu Z, Weiss A. Negative regulation of CD45 by differential homo- immunoglobulin-like receptor-A in mast cell line by associating with dimerization of the alternatively spliced isoforms. Nat Immunol 2002; subunits common to Fc receptors. JBiolChem1999; 273: 30288–96. 3: 764–71. 32 Batuwangala T, Leduc M, Gibbins JM, Bon C, Jones EY. Structure of 22 Hansen JL, Sheikh SP. Functional consequences of 7TM receptor the snake-venom toxin convulxin. Acta Crystallogr D Biol Crystallogr dimerization. Eur J Pharm Sci 2004; 23: 301–17. 2004; 60: 46–53.

2007 International Society on Thrombosis and Haemostasis