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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institut National de la Santé
et de la Recherche Médicale (INSERM) E9907, Faculté Xavier
Bichat, Paris, France; INSERM U 362, Institut Gustave Roussy,
Villejuif, France; and Millennium Pharmaceuticals Inc, Cambridge, MA.
Injuries to the vessel wall and subsequent exposure of collagen
from the subendothelial matrix result in thrombus formation. In
physiological conditions, the platelet plug limits blood loss. However,
in pathologic conditions, such as rupture of atherosclerotic plaques,
platelet-collagen interactions are associated with cardiovascular and
cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a
crucial role in collagen-induced activation and aggregation of
platelets, and people who are deficient in GPVI suffer from bleeding
disorders. Based on the fact that GPVI is coupled to the Fc
receptor (FcR)- Glycoprotein VI (GPVI) is a 58-kd (62 kd after
reduction) platelet membrane glycoprotein that plays a crucial role in
the collagen-induced activation and aggregation of
platelets.1,2 Upon injury to the vessel wall and
subsequent damage to the endothelial lining, exposure of the
subendothelial matrix to the blood flow results in deposition of
platelets. Collagen fibers are the most thrombogenic macromolecular
components of the extracellular matrix, with collagen types I, III, and
VI being the major forms found in blood vessels. Platelet interaction
with collagen occurs as a 2-step procedure: (1) the initial adhesion to
collagen is followed by (2) an activation step leading to platelet
secretion, recruitment of additional platelets, and
aggregation.3,4 In physiologic conditions the resulting
platelet plug is the initial hemostatic event limiting blood loss.
However, platelet-collagen interactions may also have major pathologic
consequences. Exposure of collagen after rupture of atherosclerotic
plaques is a major stimulus of thrombus formation associated with
myocardial infarction or stroke.
The mechanism of collagen-platelet interactions is complex. It
involves direct or indirect binding of collagen to several platelet
receptors including the GPIb-IX-V complex, integrin Collagen, collagen-related peptides (CRPs), and a snake venom protein,
convulxin (Cvx), activate platelets similar to anti-GPVI Fab'-2
fragments via GPVI-coupled signaling pathway.9-11
Protein-tyrosine phosphorylation is a key signaling event in this
pathway. GPVI is constitutively associated with the Fc receptor- Due to the strong similarity between GPVI and the multimeric Fc
receptors, mouse and human megakaryocyte libraries were examined for
the presence of an FcR-like complementary DNA (cDNA). A cDNA clone with
these characteristics was identified in both murine and human
megakaryocyte libraries. Our results demonstrate that these clones
encode for GPVI.
Recently, another group also reported the cloning of human
GPVI.22 Commensurate with their results, we show that GPVI
belongs to the Ig superfamily. In addition, we have isolated the murine homologue of GPVI and have shown that expression of both human and
mouse GPVI is restricted to the megakaryocyte/platelet lineage. We also
present considerable functional evidence including its association with the FcR- Library construction
Primary mouse megakaryocytes were obtained from day-15 fetal
livers, cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco), 10% fetal calf serum (FCS), and PEG-rHuMGDF for 4 days as
previously described.24 cDNA libraries of both human and mouse megakaryocytes were constructed using a previously
described method.25 Random EST sequences of these
libraries were searched using Basic Local Alignment Search Tool (BLAST)
algorithms to find novel membrane-bound proteins.
Chromosomal mapping
For human mapping, the primers were designed from the 3'-UTR (untranslated region) of the sequence: forward primer 5'-ACTCAAAGACTGGCGTGTGTG-3' and reverse primer 5'-GTTCTGAGAGACGAAAGGAG-3'. The PCR amplification conditions were the same as those used for the mouse mapping. The products were amplified from the G4 Radiation Hybrid Panel (Research Genetics, Huntsville, AL), run on a 2% agarose gel, and individually scored as to the presence or absence of the human band in each of the 93 cell line DNAs in addition to a negative water control. The results were analyzed with the MapManager software program. Protein preparation The open-reading frame of the predicted extracellular domain of T268/GPVI was PCR-amplified from the Kozak sequence before the first methionine to asparagine 269, immediately prior to the predicted transmembrane sequence. The PCR fragment was ligated into a pCDM8 host vector containing the genomic sequence of the human IgG1 Fc domain, such that the extracellular part of the hGPVI cDNA was fused at its C-terminus via a 3 alanine linker to the hFc sequence. The sequenced DNA construct was transiently transfected into HEK 293T cells in 150-mm plates using Lipofectamine (Gibco) according to the manufacturer's protocol. After 72 hours of transfection, the serum-free conditioned medium (OptiMEM, Gibco) was harvested, spun, and filtered. The cells were refed with fresh medium and harvested as above for another 72 hours. Using an antihuman IgG Fc polyclonal antibody, analysis of supernatants on Western blot after reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant amounts of the recombinant human soluble GPVI fusion (rhusGPVI:Fc) protein in the supernatants, with a relative molecular mass of approximately 75-80 kd relative to the Mark 12 molecular weight standards cocktail (Novex, San Diego, CA).The conditioned media was passed over a 10-mL Prosep-G protein G column
(Bioprocessing, Princeton, NJ). The column was then washed with
phosphate-buffered saline (PBS) (pH 7.4) and eluted with 200 mmol/L
glycine (pH 3.0) at 7 mL/min. Fractions from the 280-nm elution peak
containing rhusGPVI:Fc were pooled and dialyzed in 8000 MWCO dialysis
tubing against 2 changes of 4 liters PBS (pH 7.4) at 4°C with
constant stirring. The buffered exchanged material (0.2 µm) was then
sterile filtered (Millipore, Bedford, MA) and frozen at In situ hybridization In situ hybridization (ISH) was performed with day-12.5 C57BL/B6 mouse embryos and normal 4- to 6-week-old C57BL/6 mouse femurs. The tissues were fixed in 10% formalin, paraffin embedded, and subsequently sectioned at 4 µm onto Superfrost plus slides. The femurs were decalcified in TBD-2 (Shandon, Pittsburgh, PA) prior to paraffin embedding. The sections were deparaffinized in xylene, hydrated through a series of graded ethanol washes, and placed in diethyl pyrocarbonate (DEPC)-treated PBS (pH 7.4) before being processed for ISH. The sections were incubated in 20 µg/mL proteinase K (Sigma) in DEPC-PBS for 15 minutes at 37°C and then immersed in 4% formaldehyde/PBS for 5 minutes.The sections were treated with 0.2 N HCl for 10 minutes followed by DEPC-PBS. The sections were then rinsed in 0.1 mol/L triethanolamine HCl (TEA, pH 8.0), incubated in 0.25% acetic anhydride-TEA for 10 minutes, rinsed in DEPC-PBS, dehydrated through a series of graded ethanol washes, and air dried. Labeling and hybridization of sulfur 35 (35S)-radio-labeled 2.5 × 107 cpm/mL cRNA antisense and sense RNA probes encoding a 599-bp fragment of the 5'-end of the GPVI gene (generated with the PCR primers forward 5'-CAGCCTCACCCACTTTCTTC-3', nucleotides [nt] 8-27, and reverse 5'-CCACAAGCACTAGAGGGTCA-3', nt 607-588) were performed as previously described.27 Following hybridization, the sections were dehydrated rapidly through serial ethanol and 0.3 mol/L sodium acetate before being air dried, dipped in a nuclear track emulsion (NTB-2; Eastman Kodak, Rochester, NY), and exposed for 60 days at room temperature. The slides were developed with D-19 (Eastman Kodak), stained with hematoxylin and eosin-Y, and added to cover slips. Cell lines The HEL (erythroid/MK), U937 (monoblast), K562 (erythroid), CEM (T cell), HEPG2, and Hela cell lines (American Type Culture Collection [ATCC], Manassas, VA) and the FDC-P1 and 32D cell lines (D. Metcalf, The Walter and Eliza Hall Institute, Melbourne, Australia) were used in this study. The UT7 (erythroid/MK) transduced by c-mpl,28 TF1 (erythroid), KG1 (myeloblast), HL60 (myeloblast/promyelocyte), MO-7E (MK), Meg-01 (MK), and DAMI (MK) were obtained from the different laboratories that derived them.29-35The HEL, U937 HL60, Meg-01, KG1, and K562 human cell lines were cultured in IMDM with 10% FCS (Stem Cell Technology, Vancouver, British Columbia, Canada). The c-mpl UT7, TF1, and MO-7E are factor-dependent and were grown in the presence of either 2 ng/mL granulocyte macrophage(GM)-CSF or 10 ng/mL PEG-rHuMGDF in IMDM with 10% FCS. CEM and Hela were grown in Roswell Park Memorial Institute medium (RPMI) (Gibco). FDC-P1, 32D, and Ba/F3 murine cell lines were cultured in DMEM with 10% FCS (Stem Cell Technology). The cultures were performed at 37°C in a fully humidified atmosphere of 5% carbon dioxide (CO2). Samples Human megakaryocytes were obtained as described for the human libraries from mobilized or cord blood CD34+. A fetal liver was obtained following abortion at 12-week gestation after obtaining informed consent.Northern blot/reverse transcriptase-PCR analysis Human multiple tissue Northern blots (Clontech, Palo Alto, CA) were hybridized to a 1.0-kb human GPVI probe as described by the manufacturer. Total RNA was isolated using RNA PLUS (Bioprobe Systems, France), a modification of the acid-guanidinium thiocyanate-phenylchloroform extraction method of Chomczynski et Sacchi.36 RNA was reverse transcribed with random hexamers using SUPERSCRIPT reverse transcriptase (RT) (Gibco BRL/Life Technologies, Cergy Pontoise, France).For human cell lines and tissues, after reverse transcription, each
sample was subjected to a specific amplification of GPVI and
GPVI-expressing cell lines Chinese hamster ovary (CHO) cells were transfected using lipofectamine (Gibco) according to manufacturer's instructions. Full-length GPVI cDNA was cloned into an expression vector driven by an SRalpha promoter. Control CHO cells were transfected with the empty vector. The cells were collected 2 days after transfection and lysed in 12 mmol/L Tris (tris[hydroxymethyl] aminomethane), 300 mmol/L sodium chloride (NaCl), and 12 mmol/L EDTA (ethylenediamine tetraacetic acid) containing 2 µmol/L leupeptin, 2 mmol/L PMSF (phenylmethylsulfonyl fluoride), 5 KIU aprotinin, and 0.2% (vol/vol) NP40 (Sigma). After 20 minutes at 4°C under agitation, the samples were centrifuged at 13 000g for 15 minutes at 4°C, and the supernatants were frozen at 80°C for analysis.
The human cell lines HEL, U937, and K562 and the murine cell lines
FDC-P1, 32D, and Ba/F3 were engineered37 to express GPVI using the pMSCVpac retrovirus.38 Briefly, viruses carrying
the full-length cDNA encoding human or murine GPVI were constructed using base-perfect PCR-amplified fragments of the cDNAs (Clontech Laboratories, Palo Alto, CA). Viral supernatants were generated into
the 293-EBNA cells (Invitrogen, Carlsbad, CA) by transfecting the
retroviral construct and 2 pN8- Platelet preparation Blood from healthy volunteers was collected by venipuncture on acid-citrate-dextrose anticoagulant (ACD-A). When needed, the platelets were labeled by incubating the platelet-rich plasma (PRP) with 0.6 µmol/L carbon 14 5-hydroxytryptamine (14C 5-HT) at 37°C for 30 minutes. The platelet pellets were obtained by centrifugation of PRP and were washed 2 times as previously described.10Platelet aggregation and secretion Aggregation of washed platelets (3 × 108/mL) in reaction buffer was initiated by collagen type I (Bio/Data, Horsham, PA) or Cvx. The experiments were performed with stirring at 37°C in an aggregometer (Chrono-Log, Haverton, PA). Release of 14C 5-HT was measured as described previously.10Cvx preparation Cvx was purified from the venom of Crotalus durissus terrificus mainly as described by Francischetti et al,39 using a 2-step gel filtration procedure of sephadex G75 (Pharmacia Biotech, Uppsala, Sweden) followed by sephacryl S100 (Pharmacia Biotech). Cvx was labeled with iodine 125 (125I) using the iodogen procedure (Pierce Chemical, Rockford, IL) and Na-125I (Amersham, Les Ulis, France). Cvx was coupled to fluorescein isothiocyanate (FITC) by mixing Cvx in 50 mmol/L NaHCO3 and 150 mmol/L NaCl (pH 9.5) with a 100-fold molar excess of FITC (Aldrich, St Quentin Fallavier, France) at 4°C overnight. FITC-coupled Cvx was separated from free FITC by chromatography on a sephadex G25 column in 20 mmol/L phosphate and 150 mmol/L NaCl (pH 7.4) in PBS. Bothrojaracin, a specific thrombin inhibitor purified from the venom of Bothrops jararaca as previously described,40 was coupled to FITC using the same procedure.Flow cytometry Cells transduced with human or murine GPVI viruses or the control virus were incubated in the presence of 20 nmol/L FITC-Cvx or FITC-bothrojaracin for 60 minutes at room temperature. After dilution in PBS, the cells were analyzed by a fluorescence-activated cell sorter (FACS) flow cytometer (FACSort; Becton Dickinson, Franklin Lakes, NJ).Protein analysis Platelets, megakaryocytes, and cell lines were lysed in a buffer composed of 12 mmol/L Tris, 300 mmol/L NaCl, and 12 mmol/L EDTA containing 2 µmol/L leupeptin, 2 mmol/L PMSF, 5 KIU aprotinin, and 0.2% (vol/vol) NP40. After 20 minutes at 4°C under agitation, the samples were centrifuged at 13 000g for 15 minutes at 4°C, and the supernatants were frozen at80°C. The protein
concentration was determined using a protein assay (Bio-Rad
Laboratories, Ivry-sur-Seine, France). For blotting experiments, the
proteins were further solubilized with 2% SDS at 100°C for 5 minutes. The proteins were separated by electrophoresis on acrylamide
slab gels (Mini Protean II, Bio-Rad) and transferred on a
polyvinylidene fluoride (PVDF) membrane. The membranes were soaked with
5% nonfat dry milk and incubated with either 6 × 103
Bq/mL 125I-Cvx in PBS (pH 7.4) containing 0.1% (vol/vol)
Tween 20, or with 9 µg/mL anti-GPVI IgG in PBS (pH 8) containing
0.02% (vol/vol) Tween 20 in the absence or presence of 0.5 µmol/L
cold Cvx. Anti-GPVI IgG was obtained as previously
described10 from patient plasma (gift of Prof M. Okuma, Kyoto, Japan).
The antibodies were revealed using peroxidase-coupled protein A and
enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala,
Sweden). For immunoprecipitation, cell lysates were precleared by
incubation with protein A-sepharose at 4°C for 30 minutes followed by
centrifugation. Cleared lysates were incubated overnight at 4°C with
10 µg/mL polyclonal anti-FcR- Cell adhesion Collagen type I (2 µg) (Chrono-Log), 1.4 µg Cvx, or 2 µg BSA (Sigma) in 100 µL PBS were immobilized on Immulon II plates (Dynatech, St Cloud, France) at 4°C overnight. The plates were then saturated with 2 mg/mL BSA in PBS for 1 hour and washed with PBS. The cells in culture medium were labeled with chromium 51 (51Cr) (CIS Bio International, Gif sur Yvette, France) at 37°C for 1 hour after centrifugation at 150g for 10 minutes. The cells were washed with Hank's balanced salt solution containing 2 mg/mL BSA and resuspended in the same buffer. The cells were added to the wells. After 60 minutes at room temperature, the wells were emptied and washed, and the samples were counted for 51Cr.
Cloning of GPVI Using computer-based searching of EST sequences from a murine megakaryocyte library, we isolated a clone, named T268, that showed striking homology to the inhibitory family of receptors. The human homologue was identified from a human megakaryocyte library by sequence homology. These clones encode proteins of amino acids 313 and 339, respectively, which are predicted to be type I membrane proteins belonging to the Ig superfamily. Alignment of the mouse and human T268 sequences shows 64.4% conservation in amino acids between species (Figure 1A). Alignment of the mouse and human T268 nucleotide sequences shows 67.3 % identity (Figure 1B). The closest database matches for these proteins are the natural killer (NK) inhibitory receptors, although it is clear that there are no inhibitory or activation motifs present in the relatively short cytoplasmic domain. The extracellular domain contains 2 Ig-like C2 domains and a conserved N-linked glycosylation site at asparagine 93. A second potential glycosylation site is also found at asparagine 244 in the mouse protein. It is interesting to note the presence of a charged residue in the transmembrane domain (R273 in hT268 and R270 in mT268), which may act as an interaction site for association with other membrane proteins.
These characteristics of human GPVI are identical to those recently
reported by Clemetson et al.22 The mouse gene for GPVI was
mapped on radiation hybrid panels to chromosome 7, between the flanking
markers D7Mit152 and D7Mit178. The human gene was localized to the long
arm of human chromosome 19 (19q), 11.1cR distal to genetic marker
WI-5264 and 19.3cR proximal to genetic marker WI-5423. This region is
equivalent to 19q13.32-q13.33 (region 3, band 2 to band 3). This region
contains the melanoma inhibitory activity gene and is part of the
leukocyte receptor complex (LRC) that contains various members of the
Ig superfamily. The closest mapped genes to GPVI included the FcR- Tissue expression of GPVI To determine tissue distribution of both mouse and human GPVI, Northern blot, RT-PCR, and ISH were performed. Human tissues were studied using Northern blot or RT-PCR analysis. Northern blots (Figure 2A) revealed no specific message in samples of brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, or lymph node. A 2-kb transcript was observed only in bone marrow and fetal liver. A signal was inconsistently observed with peripheral blood cells, probably due to platelet RNA contamination in some samples. Indeed, transcripts for platelet GPIIb, a platelet specific protein, were also detected in these positive samples (data not shown).
Using RT-PCR analysis (Figure 2B), the GPVI transcript was detected only in platelets, not the other blood cells. In cell lines, a strong PCR signal was observed in the HEL, MEG01, DAMI, TPO-stimulated MO7E, and mpl-transduced UT7 cell lines.28 A very low signal was also detected in the K562 and KG1 cell lines, 2 cell lines that also express GPIIb at a low level. But there was no expression observed in the HEPG2, CEMT, TF1, U937, HL60, and Hela cells. CD41+ cells (including more than 95% megakaryocytes) isolated from normal cord blood or chemotherapy-induced mobilized peripheral blood displayed a strong RT-PCR signal. Fetal liver cells expressed a moderate level of expression compared to megakaryocyte-enriched samples. Mouse tissues were studied using Northern blot and ISH analysis. ISH
reveals that GPVI was exclusively found in the liver during
embryogenesis (Figure 3A,B). The signal
pattern was strong and multifocal, suggestive of expression by a
scattered cell population. This signal was observed at embryonic day
13.5, 14.5, and 16.5 and decreased in intensity at day 18.5 and in a
1.5-day-old newborn (data not shown). In adults, the expression in
liver was no longer observed, but a strong multifocal signal was seen
in spleen and bone marrow. There was no signal observed in any other
tissues including samples of brain, eye, harderian gland, submandibular gland, bladder, white fat, stomach, brown fat, heart, adrenal gland,
colon, small intestine, liver, placenta, thymus, lymph node, lung,
spinal cord, pancreas, skeletal muscle, and testes. Photoemulsion
processing of the spleen and bone marrow showed that this expression
was restricted to megakaryocytes (Figure 3C).
In conclusion, despite screening a large number of human and mouse tissues, GPVI expression was detected only in megakaryocytes and platelets. This result strongly suggests that GPVI is restricted to this hematopoietic lineage. Identification of recombinant GPVI Cvx, a very high-affinity specific ligand of GPVI, was used to detect the expression of recombinant GPVI. The CHO cells transfected with the empty vector or the vector containing human T268 cDNA were tested for 125I-Cvx binding in ligand blotting experiments. 125I-Cvx clearly labeled a single band in CHO cells only transfected with the vector containing human T268 cDNA (Figure 4), which strongly suggested that recombinant GPVI is encoded by T268. The migration of the hypothetical recombinant GPVI is slightly faster than that of platelet GPVI (approximately 54 kd vs approximately 58 kd) but very close to that of megakaryocyte GPVI (approximately 55 kd). GPVI production by CHO cells was confirmed by Western blot with human anti-GPVI antibodies (Figure 4B). In platelets, a 58-kd band corresponding to GPVI and a high molecular weight band corresponding to platelet IgG revealed by protein A were observed. Several proteins were nonspecifically labeled in CHO, but a clear 54-kd band was observed exclusively in lysats from T268-transfected CHO. Proof that Cvx and anti-GPVI bind to the same protein is provided by the observation that Cvx inhibits binding of anti-GPVI to recombinant GPVI as well as to platelet GPVI (Figure 4C), indicating that anti-GPVI and Cvx compete for binding to the corresponding protein. Together, these results demonstrate that the protein encoded by T268 is recombinant GPVI. The difference between the molecular mass of platelet GPVI and recombinant GPVI is most probably due to an incomplete or differential processing of the protein in CHO cells compared with platelets or megakaryocytes.
Functional studies Flow cytometry.
To determine whether recombinant GPVI was expressed at the cell
surface, different human or murine hematopoietic cell lines were
transduced with recombinant retroviruses expressing human or murine
GPVI and with the control retrovirus. We observed that the cell lines
used for this study express FcR-
Cell adhesion.
Because GPVI appeared to be expressed at the cell surface of
transduced cells, we tested its capacity to promote cell adhesion in a
static system, to either immobilized Cvx or collagen, and then compared
this to immobilized BSA. We tested 2 cell lines: U937 and FDC-P1.
Neither the cells expressing GPVI nor the control cells bound to
immobilized BSA. However, expression of recombinant human or mouse GPVI
in U937 or FDCP-1, respectively, clearly promotes the adhesion of these
cells to immobilized collagen and to a greater extent to immobilized
Cvx (Figure 6).
Association of recombinant GPVI with FcR-  chain, we performed immunoprecipitation studies with an anti-FcR- polyclonal antibody on GPVI-transduced U937 cell
lysates. Figure 7 shows the precipitated
proteins analyzed by immunoblotting with a mixture of anti-FcR- and
anti-GPVI antibodies. We observed 3 bands in all samples: a high
molecular weight band corresponding to IgG, an approximately 50-kd
nonidentified band, and a 14-kd doublet corresponding to the FcR-
chain. In addition, 1 band corresponding to GPVI is present in
platelets and also in U937 transduced with the GPVI virus, but it is
not present in U937 transduced with the control virus. This indicates
that recombinant GPVI is associated with the FcR-
chain.
Inhibition of collagen and Cvx-induced platelet activation by rhusGPVI:Fc We produced and purified the rhusGPVI:Fc protein to investigate its ability to compete with membrane-bound platelet GPVI. The RhusGPVI:Fc protein is composed of 2 amino acid fragments (1-269) of GPVI linked by their C-terminus to a human IgG1 Fc domain, has a molecular mass of 75-80 kd, and is divalent. The rhusGPVI:Fc protein did not induce platelet aggregation or granule secretion alone. When 0.25-5 µg/mL rhusGPVI:Fc was incubated with Cvx prior to the addition of platelets, aggregation and dense granule secretion was fully inhibited (Figure 8A). In addition, when 1 µg/mL rhusGPVI:Fc was added to the platelet suspension prior to Cvx, it also inhibited aggregation and secretion, indicating that it could compete with membrane-bound platelet GPVI for Cvx (Figure 8A).
When the agonist was collagen, incubation of rhusGPVI:Fc with collagen prior the addition of platelets inhibited platelet aggregation and secretion (Figure 8B). This inhibition was dose-dependent, with a 10-fold higher concentration of rhusGPVI:Fc (2.5 µg/mL) needed to reach a full inhibitory effect than that required for Cvx. Furthermore, when up to 5 µg rhusGPVI:Fc was added to platelets prior to the collagen, no inhibition was observed (Figure 8B).
GPVI has long been recognized as an important receptor for
platelet function. Identification of its structure and characterization of its expression are important steps in clarifying its role in thrombosis. Based on the hypothesis that GPVI should share structural similarities with the Fc receptors coupled to the As predicted, GPVI belongs to the Ig superfamily of cell surface
molecules. It is a type I membrane protein containing 2 extracellular Ig-like domains. The closest relatives of GPVI in the public databases are the growing family of inhibitory receptors, although this relationship is due solely to similarities in the extracellular domain.
The sequence of our human clone is identical to that recently reported
by Clemetson et al.22 The homology between mouse and human
GPVI is approximately 64%. This homology is spread throughout the
molecule, but is slightly higher (78%) in the Ig-like domains. Interestingly, both human and mouse GPVI contain conserved variants of
the WSXWS box (residues 97-101 and 192-196). This motif is a signature
of class I hematopoietic receptors, but variants are also found in the
sequences of all killer-cell inhibitory receptors (KIRs).41 These motifs have been shown to contribute to
tertiary folding. GPVI has a relatively short cytoplasmic tail with no obvious signaling motifs analogous to the ITAMs and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of other signaling receptors. GPVI has a positively charged residue in the transmembrane domain that
should allow it to form complexes with the FcR- Human GPVI was mapped to 19q13.32-q13.33. Multiple members of the Ig
superfamily, including KIRs, Ig-like transcripts (ILT-1, -2, and -3),
the gp49b family, and FcR- The tissue distribution of GPVI is currently poorly defined. To
determine the expression pattern of GPVI, we examined an extensive panel of human and mouse tissues. The only cells where we could detect
a significant amount of GPVI RNA were megakaryocytes and platelets.
Furthermore, by searching internal and public gene databases, the only
cDNA sources that were found were various Millennium megakaryocyte
libraries. These results strongly suggest that the expression of GPVI
is highly specific to the megakaryocytic lineage. It remains to be
determined if GPVI expression is regulated throughout megakaryopoiesis.
Presently, there are very few molecules that are specific to the
megakaryocyte lineage. GPIIb (integrin Functional characterization of recombinant GPVI was performed using
transfected cells that have either no levels of endogenous GPVI (U937
and FDC-P1) or low levels (HEL). In contrast to the work described by
Clemetson et al,22 who used DAMI cells that expressed GPVI
messenger RNA, this characterization allowed us to study GPVI
functional properties independently of endogenous GPVI. Our extensive
analysis showed that the recombinant protein GPVI was based on binding
to Cvx, binding to collagen, binding of anti-GPVI IgG isolated from a
patient, and association to the FcR- Cvx, a snake venom protein, has been shown to bind specifically to platelet GPVI.10,11,17,39 In ligand binding studies we demonstrated that Cvx bound to CHO cells that transiently expressed the cloned genes. The observed molecular weight of GPVI in these cells was lower than that attributed to GPVI in platelets but similar to that observed in primary megakaryocytes. This difference in migration may be due to differences in glycosylation, and it is possible that this is dependent on the stage of cell differentiation. Indeed, GPVI appears to be heavily glycosylated. The predicted molecular weight based on the cDNA sequence is around 38 kd, but when the molecular weight is deduced from SDS-PAGE under nonreducing conditions, the molecular weight is 54 kd in CHO cells and 58 kd in platelets. This is consistent with glycosylation and accounts for approximately one-third of the mass of the protein. Human GPVI has only 1 N-linked glycosylation site, which appears to account for only 3 kd (data not shown). It is likely that the majority of glycosylation is due to O-linked carbohydrates on the large clusters of serine and threonine residues present in the extracellular domain. Using ligand-binding fluorescence analysis, we showed that Cvx binds to
the human recombinant protein in U937 and K562 cells and to the mouse
recombinant protein in FDC-P1, 32D, and Ba/F3. It is known that Cvx
recognized mouse GPVI from previous studies showing that Cvx is a
potent platelet activator of both human and mouse
platelets.17 The expression of recombinant GPVI at the
cell surface may have been facilitated by the coexpression of the
FcR- The importance of GPVI in collagen-induced platelet activation was discovered following the characterization of a patient suffering from immune thrombocytopenic purpura.8 This patient developed self-antibodies that recognized GPVI and induced platelet aggregation. In contrast, Fab fragments do not induce platelet activation and instead inhibit the aggregation induced by collagen. In this paper we show that antibodies from this patient recognize the protein in CHO cells expressing our clone. Interestingly, Cvx inhibited the binding of anti-GPVI antibodies to the recombinant protein, as previously observed on platelets. GPVI, despite its essential role in collagen-induced platelet
aggregation, is described as having a minor role in platelet adhesion
to collagen. Other receptors, such as the GPIb-IX-V complex or the
integrin The difference in reactivity between collagen and Cvx is further
emphasized by the differences in the inhibitory effect that the soluble
GPVI protein has on collagen- and Cvx-induced platelet activation.
Indeed, soluble GPVI inhibits Cvx-induced platelet activation in the
absence of preincubation with Cvx, while it requires a preincubation
with collagen to inhibit collagen-induced platelet activation. This
probably reflects the rapid kinetics of interaction between GPVI and
Cvx compared to those between GPVI and collagen. The affinity of
soluble GPVI for Cvx is probably very high for 2 reasons: (1) soluble
GPVI is expressed in a divalent Fc fusion form, and (2) Cvx is
multivalent due to its trimeric GPVI may play an important role in the development of thrombi because
it is the receptor that appears to govern platelet activation at the
contact of collagen and thus induces platelet recruitment. Indeed,
patients with GPVI deficiency, whether it was associated or not
associated with an anti-GPVI antibody, displayed bleeding disorders.7,8 The molecular cloning of GPVI provides the opportunity to characterize the mechanism of these deficiencies, not
only the precise interaction between GPVI and the integrin Even if GPIIb-IIIa were involved in platelet adhesion, its principal role is to bind fibrinogen, thereby allowing platelet aggregation and serving as the final common pathway of platelet thrombus formation regardless of the metabolic pathway initiating platelet activation. In contrast, GPVI is involved in an early step of platelet activation occurring immediately when platelets contact the subendothelial matrix. GPVI may represent an alternative and more specific target for new antithrombotic compounds. The antagonist can be directed against either of the 2 players, ie, collagen GPVI binding sites or GPVI itself. Because our observations suggest that the GPVI binding sites are not easily accessible on collagen fibers, one might speculate that an antagonist directed against GPVI may be more efficient than an antagonist directed against collagen. Altogether, our results provide evidence that a new member of the Ig superfamily is platelet GPVI, a collagen receptor specific for the megakaryocyte lineage. GPVI characterization allows us to get further insight into its role as collagen receptor, and forthcoming studies will explore its involvement in thrombotic disease, bleeding disorders, and megakaryocyte maturation.
The authors are grateful to Prof Stuart Orkin and Dr Paresh Vyas (Children's Hospital, Boston, MA), Dr David Gearing, Dr Charles Gray, and R. Clark (Millennium Pharmaceuticals, Cambridge, MA) for their contribution to the megakaryocyte library constructions; Prof M. Okuma (Kyoto University, Kyoto, Japan) for the anti-GPVI patient plasma; Dr Ivo M. B. Francischetti (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for the generous gift of Crotalus durissus terrificus venom; Christopher Comrack, Belle Chang, Thao Duong, and Kevin McDonald for excellent technical assistance; and Amgen (Thousand Oaks, CA) for providing recombinant PEG-HuMGDF and hSCF.
Submitted January 27, 2000; accepted April 27, 2000.
Supported by grants from the University Paris 7, Paris, France, and grant 9472 from the Association de Recherche contre le Cancer, Villejuif, France.
M.J.-P. and S.B. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jean-Luc Villeval, Inflammation, Millennium Pharmaceuticals Inc, 45 Sydney St, Cambridge MA 02139; e-mail: villeval{at}mpi.com.
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D. A. Wong, Y. Kita, N. Uozumi, and T. Shimizu Discrete Role for Cytosolic Phospholipase A2{alpha} in Platelets: Studies Using Single and Double Mutant Mice of Cytosolic and Group IIA Secretory Phospholipase A2 J. Exp. Med., August 5, 2002; 196(3): 349 - 357. [Abstract] [Full Text] [PDF]
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F. Krotz, H. Y. Sohn, T. Gloe, S. Zahler, T. Riexinger, T. M. Schiele, B. F. Becker, K. Theisen, V. Klauss, and U. Pohl NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment Blood, July 18, 2002; 100(3): 917 - 924. [Abstract] [Full Text] [PDF]
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K. Suzuki-Inoue, D. Tulasne, Y. Shen, T. Bori-Sanz, O. Inoue, S. M. Jung, M. Moroi, R. K. Andrews, M. C. Berndt, and S. P. Watson Association of Fyn and Lyn with the Proline-rich Domain of Glycoprotein VI Regulates Intracellular Signaling J. Biol. Chem., June 7, 2002; 277(24): 21561 - 21566. [Abstract] [Full Text] [PDF]
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D. Locke, H. Chen, Y. Liu, C. Liu, and M. L. Kahn Lipid Rafts Orchestrate Signaling by the Platelet Receptor Glycoprotein VI J. Biol. Chem., May 17, 2002; 277(21): 18801 - 18809. [Abstract] [Full Text] [PDF]
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T. M. Quinton, F. Ozdener, C. Dangelmaier, J. L. Daniel, and S. P. Kunapuli Glycoprotein VI-mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP Blood, May 1, 2002; 99(9): 3228 - 3234. [Abstract] [Full Text] [PDF]
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K. Furihata, K. J. Clemetson, H. Deguchi, and T. J. Kunicki Variation in Human Platelet Glycoprotein VI Content Modulates Glycoprotein VI-Specific Prothrombinase Activity Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1857 - 1863. [Abstract] [Full Text] [PDF]
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 S. A. Croft, N. J. Samani, M. D. Teare, K. K. Hampton, R. P. Steeds, K. S. Channer, and M. E. Daly Novel Platelet Membrane Glycoprotein VI Dimorphism Is a Risk Factor for Myocardial Infarction Circulation, September 25, 2001; 104(13): 1459 - 1463. [Abstract] [Full Text] [PDF]
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K. L. Jones, S. C. Hughan, S. M. Dopheide, R. W. Farndale, S. P. Jackson, and D. E. Jackson Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions Blood, September 1, 2001; 98(5): 1456 - 1463. [Abstract] [Full Text] [PDF]
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S. Moog, P. Mangin, N. Lenain, C. Strassel, C. Ravanat, S. Schuhler, M. Freund, M. Santer, M. Kahn, B. Nieswandt, et al. Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation Blood, August 15, 2001; 98(4): 1038 - 1046. [Abstract] [Full Text] [PDF]
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B. Nieswandt, W. Bergmeier, A. Eckly, V. Schulte, P. Ohlmann, J.-P. Cazenave, H. Zirngibl, S. Offermanns, and C. Gachet Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation Blood, June 15, 2001; 97(12): 3829 - 3835. [Abstract] [Full Text] [PDF]
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B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.-P. Cazenave, P. Ohlmann, C. Gachet, and H. Zirngibl Long-Term Antithrombotic Protection by in Vivo Depletion of Platelet Glycoprotein VI in Mice J. Exp. Med., February 19, 2001; 193(4): 459 - 470. [Abstract] [Full Text] [PDF]
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A.-H. Lagrue-Lak-Hal, N. Debili, G. Kingbury, C. Lecut, J.-P. Le Couedic, J.-L. Villeval, M. Jandrot-Perrus, and W. Vainchenker Expression and Function of the Collagen Receptor GPVI during Megakaryocyte Maturation J. Biol. Chem., April 27, 2001; 276(18): 15316 - 15325. [Abstract] [Full Text] [PDF]
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Y.-M. Zheng, C. Liu, H. Chen, D. Locke, J. C. Ryan, and M. L. Kahn Expression of the Platelet Receptor GPVI Confers Signaling via the Fc Receptor gamma -Chain in Response to the Snake Venom Convulxin but Not to Collagen J. Biol. Chem., April 13, 2001; 276(16): 12999 - 13006. [Abstract] [Full Text] [PDF]
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W. Bergmeier, D. Bouvard, J. A. Eble, R. Mokhtari-Nejad, V. Schulte, H. Zirngibl, C. Brakebusch, R. Fassler, and B. Nieswandt Rhodocytin (Aggretin) Activates Platelets Lacking alpha 2beta 1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibalpha J. Biol. Chem., June 29, 2001; 276(27): 25121 - 25126. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2
1, GPIV, GPVI, and 65- and 85-kd proteins.
/RI, and Fc-
