Introduction

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ADP plays a key role in hemostasis by acting as an aggregating agent. In addition, through its secretion from platelet dense granules, it contributes to and reinforces the platelet aggregation induced by other agents such as collagen or thrombin (Mills, 1996; Gachet, 2001). Stimulation of platelets with ADP leads to an increase in intracellular calcium through mobilization of internal calcium stores, rapid calcium entry from the external medium and inhibition of adenylyl cyclase activity (Mills, 1996; Gachet, 2001). All these effects are caused by the interaction of ADP with specific P2 receptors that have been characterized over the past 3 years (Gachet, 2001). The metabotropic P2Y₁ receptor is expressed in a wide range of body tissues (Ralevic and Burnstock, 1998) and in blood platelets (Leon et al., 1997). This entity, initially described as a receptor for which ATP was an agonist (Schachter et al., 1996), was later demonstrated to be, in fact, an ADP receptor (Leon et al., 1997; Hechler et al., 1998c). Additional studies have demonstrated that the effect of ATP on the P2Y₁ receptor depends upon receptor density [i.e., at a low level of receptor abundance, ATP acts as an antagonist, whereas at higher receptor density, ATP could behave as a partial agonist (Palmer et al., 1998)]. Pharmacological studies, using selective P2Y₁ antagonists (Hechler et al., 1998b; Jin et al., 1998a), and investigations in P2Y₁ knock-out mice (Fabre et al., 1999; Leon et al., 1999) showed this receptor to be necessary for the initiation of platelet aggregation in response to ADP through the mobilization of internal calcium stores. Studies in P2Y₁ knock-out mice also pointed to its essential role in thrombotic states (Fabre et al., 1999; Leon et al., 1999). The P2Y₁ receptor is nevertheless insufficient to support full aggregation in response to ADP, and platelets also contain the newly identified P2Y₁₂ receptor, previously called P2Ycyc, P2T_AC, P2Y_AC or P2Y_ADP (Hollopeter et al., 2001), responsible for completion of the aggregation response (Daniel et al., 1998; Hechler et al., 1998a). Finally, platelets contain a ligand-gated ion channel (MacKenzie et al., 1996; Vial et al., 1997; Scase et al., 1998; Sun et al., 1998; Takano et al., 1999), the P2X₁ receptor, which seems to play a discrete role in the aggregation induced by ADP (Takano et al., 1999). P2Y₁ mRNA has been characterized in several human leukemic cell lines with megakaryocytic features by reverse transcription-polymerase chain reaction (RT-PCR) methods (Leon et al., 1997; Jin et al., 1998b). In contrast, much less is known about the size of mouse P2Y₁ mRNA or its potential regulation by Mpl ligand.

Mpl ligand, also known as thrombopoietin or megakaryocyte growth and differentiation factor (MGDF), is the pivotal physiologic regulator of megakaryocytopoiesis and platelet production (Kaushansky, 1999). It stimulates both megakaryocyte progenitor cell proliferation and the maturation of megakaryocytes, a process involving the formation of platelet-specific granules and demarcation membranes, the expression of lineage-specific proteins, such as platelet factor 4, glycoprotein GPIIbIIIa, and the GPIb-IX-V complex and an increase in endomitosis (Kaushansky, 1999). Although Mpl ligand does not induce in vitro platelet aggregation, it enhances the effect of thrombin, collagen, or ADP on this process (Oda et al., 1996; Harker et al., 1996a; Oda et al., 1999). In other studies, platelets were derived from healthy human volunteers (Harker et al., 2000) or from nonhuman primates (Harker et al., 1996b) treated or nontreated with Mpl ligand. In each of these cases, the in vivo pretreatment with this cytokine did not affect platelet response to ADP in vitro. Because P2Y₁ is one of the key receptors of platelets, we wished to determine whether Mpl ligand could affect the expression of this receptor during megakaryocyte differentiation. In cell culture experiments, we used the mouse megakaryocytic cell line Y10/L8057, a subclone of the L8057 cell line (Ishida et al., 1993), which has been shown to respond to Mpl ligand through an increase in ploidy and specific megakaryocytic markers (Zhang et al., 1998; Thompson and Ravid, 1999). Subsequently, we examined the in vivo effects of Mpl ligand on levels of P2Y₁ receptor mRNA in spleen megakaryocytes and on platelet P2Y₁ receptor density and reactivity to ADP.

Levels of P2Y₁ receptor mRNA and protein in Y10/L8057 cells increased rapidly and significantly under Mpl ligand treatment and this effect was mediated by an increase in transcription of the P2Y₁ receptor gene. Moreover, the rise in P2Y₁ mRNA levels induced by Mpl ligand also appeared in vivo in spleen megakaryocytes, but without any repercussion on the density of the receptor on platelets or their responsiveness to ADP. This is in accordance with previous reports that Mpl ligand up-regulates GPIIbIIIa, GPIbIX, and GPV mRNA in megakaryocytes, but not the concentrations of these proteins in platelets (Harker et al., 1996b; O'Malley et al., 1996; Zauli et al., 1997; Thompson and Ravid, 1999). Hence, the Mpl ligand-induced increase in P2Y₁ receptor expression in megakaryocytes may be an integral feature of the megakaryocytic differentiation program. Because this receptor plays a key role in hemostasis and thrombosis (Gachet, 2001), the absence of enhanced platelet reactivity to ADP further suggests that clinical use of Mpl ligand to stimulate platelet production in patients with bone marrow failure might not be associated with adverse effects of platelet hyperreactivity.

	Experimental Procedures

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Materials. 2-Methylthio-adenosine 5'-diphosphate (2MeSADP) was from Sigma/RBI (Natick, MA) and human fibrinogen from Kabi (Stockholm, Sweden). ADP, adenosine 2'-phosphate 5'-phosphate (A2P5P), actinomycin D, and fatty-acid-free human serum albumin were from Sigma (St. Louis, MO). Pegylated recombinant human (or murine) MGDF [PEG-rHuMGDF (or PEG-rmMGDF)], recombinant polypeptides encompassing the amino-terminal sequence of human (or murine) Mpl ligand, were a generous gift of Amgen Inc. (Thousands Oaks, CA). Pegylation of MGDF increases its in vivo potency by roughly 10- to 20-fold, largely by delaying its clearance and thus prolonging plasma half-life (Harker et al., 1996b). AR-C69931 MX was kindly provided by AstraZeneca Charnwood (Loughborough, UK) and [³³P]2MeSADP (850 Ci/mmol) by PerkinElmer Life Sciences (Le Blanc Mesnil, France). Apyrase (adenosine 5'-triphosphate diphosphohydrolase, EC 3.6.1.5) was purified from potatoes as described previously (Hechler et al., 1998a). MRS2179 (N⁶-methyl 2'-deoxy-adenosine 3',5'-bisphosphate) was synthesized by P. Raboisson (Centre National de la Recherche Scientifique Unité Propre de Recherche 421, Faculté de Pharmacie, Illkirch, France).

Cell Culture. Y10/L8057 cells were maintained in Ham's F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (fetal calf serum, Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cultures were kept at 37°C in a humidified atmosphere containing 5% CO₂ and the cells were subcultured every 3 days to a density of 3 × 10⁵ cells/ml. In experiments using PEG-rHuMGDF, Y10 cells were cultured in Iscove's modification of Dulbecco's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum.

Northern Blot Analyses. Y10/L8057 cells were harvested and total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions, electrophoresed on a 1.2% agarose-formaldehyde gel, and transferred onto a nylon membrane. The various probes were random labeled (NEBlot kit; New England Biolabs Inc., Beverly, MA) as specified in the figure legends. Hybridization was performed at 42°C for 20 h under agitation in 5× standard saline/phosphate/EDTA, 50% formamide, 5× Denhardt's solution containing 0.5% SDS, and 100 µg/ml heat-denatured salmon sperm DNA. The membrane was washed in 2× SSC, 0.1% SDS for 15 min at 42°C, in 1× SSC for 5 min at 60°C and in 0.1× SSC, 0.1% SDS for 5 min at 60°C, before exposure to an X-ray film for up to 72 h at 80°C.

Nuclear Isolation and Run-On Assay. Nuclei were prepared from Y10/L8057 cells cultured in the absence or presence of 25 ng/ml PEG-rHuMGDF for 2 days. After isolation as described previously (Zhang et al., 1998), the nuclei were resuspended in 50 mM Tris, pH 8.3, containing 5 mM MgCl₂, 0.1 mM EDTA, and 40% glycerol at a concentration of 1 × 10⁷ nuclei/100 µl. In vitro transcription was initiated by adding 100 µl of the nuclear suspension to an equal volume of 2× reaction buffer [10 mM Tris, pH 8.0, 5 mM MgCl₂, 0.3 mM KCl, 1 mM ATP, 1 mM CTP, 1 mM GTP, 5 mM dithiothreitol, and 12.5 µl of (-³²P)UTP (10 mCi/ml)] and was allowed to proceed for 30 min at 30°C. Products of the nuclear run-on reaction were extracted into 1 ml of TRIzol according to the manufacturer's instructions. The transcribed RNA was then resuspended in 20 mM HEPES, pH 7.5, containing 5 mM EDTA and denatured by addition of 200 mM NaOH for 15 min on ice. Denaturation was stopped by addition of 250 mM HEPES (free acid). RNA was precipitated with ethanol and 3 M potassium acetate, washed in 75% ethanol and finally resuspended in 500 µl of hybridization solution (50% formamide, 5× Denhardt's solution, 0.5% SDS, 5× standard saline/phosphate/EDTA, 200 µg/ml salmon sperm DNA). pUC-19 DNA and plasmids containing P2Y₁ or GAPDH cDNA, each at 10 µg, were applied to nitrocellulose with a slot-blot apparatus. The transcription reaction mixture (10⁶ cpm/ml) was then added to the prehybridized membranes and hybridization was performed at 42°C for 24 h. After washing twice in 2× SSC, 0.1% SDS for 5 min at 42°C and once in 1× SSC, 0.1% SDS for 15 min at 60°C, the membranes were exposed to an X-ray film for up to 48 h at 80°C.

Binding of [³³P]2MeSADP to Y10/L8057 Cells and Washed Mouse Platelets. Y10/L8057 cells were washed twice by centrifugation (150 g, 20°C) in basal salt solution (25 mM HEPES, pH 7.3, 125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM glucose, and 0.1% fatty-acid-free human serum albumin) supplemented with 2 mM CaCl₂ and 0.2 U/ml apyrase (an ADP scavenger) and resuspended in basal salt solution containing calcium but no apyrase. At 20°C, a time course study of the incubation of [³³P]2MeSADP (0.6 nM, 10⁶ d.p.m.) with Y10/L8057 cells showed that a steady state was achieved after 20 min and maintained for at least 30 min. In subsequent experiments, an incubation time of 30 min was employed for convenience. The temperature of 20°C was chosen to minimize the metabolism of nucleotides by ectoenzymes, a process that would be enhanced at 37°C. Because the radioactive phosphate of [³³P]2MeSADP is in the  position, possible degradation should not interfere with binding to the P2Y₁ receptor. Thus, the binding of 2MeSADP to nontreated or PEG-rHuMGDF-treated Y10/L8057 cells was evaluated by incubation of [³³P]2MeSADP (0.6 nM, 10⁶ dpm) with Y10/L8057 cells (4 × 10⁶/ml) for 30 min at 20°C in a final volume of 0.5 ml, in the presence or absence of 1 mM A2P5P, a selective P2Y₁ receptor antagonist (Boyer et al., 1996). Similarly, the binding of 2MeSADP to washed platelets from nontreated or PEG-rmMGDF (25 µg/kg) treated mice was determined (Hechler et al., 1998a) by incubating [³³P]2MeSADP (0.5 nM, 3 × 10⁵ d.p.m.) with washed platelets in suspension (3 × 10⁵ platelets/µl) for 5 min at 37°C in a final volume of 200 µl, in the presence or absence of 1 mM A2P5P.

Preparation of Y10/L8057 Cell Membranes and Binding Studies. Y10/L8057 cells were washed twice with PBS buffer and resuspended in buffer A (10 mM HEPES, 1 mM EDTA, 5 mM MgCl₂, 20 mM PMSF, pH 7.5) at a density of 10⁷ cells/ml. After three freeze-thaw cycles, the cell suspension was homogenized with a Teflon pestle homogenizer on ice and centrifuged at 1,000g for 15 min at 4°C. The supernatant was then centrifuged at 45,000g for 15 min at 4°C; the resultant pellet was resuspended in buffer A, aliquoted, and stored at 80°C until use. Protein content was determined by the bicinchoninic acid method (Pierce, Rockford, IL), using bovine albumin as standard. Measurement of [³³P]2MeSADP binding to Y10/L8057 cell membranes (5 µg of protein/tube) was performed as described above for intact Y10/L8057 cells.

PEG-rmMGDF Injection and in Situ Hybridization Analyses of Spleen Tissues. PEG-rmMGDF diluted with 1% normal mouse serum in PBS was injected once into the lateral tail vein of 6- to 10-week-old FVB mice (50 µg/kg) as previously described (Zimmet et al., 1997). Mice injected with either PEG-rmMGDF or vehicle were sacrificed 3 days later and the spleens removed immediately and fixed in 4% paraformaldehyde overnight. Although megakaryocyte numbers and ploidy were both significantly increased, as reported elsewhere (Zimmet et al., 1997), the concentration of PEG-rmMGDF used in this protocol caused no significant change in platelet volume (Daw et al., 1998). The tissue samples were dehydrated in graded ethanol solutions, cleared with xylene, and embedded in paraffin. Sections (5 µm) were processed as described previously (Zimmet et al., 1997) before hybridization with the ³⁵S-labeled P2Y₁. Slides were stained with hematoxylin and eosin for analysis by bright field microscopy and radioactive signals were detected by autoradiography after 4 weeks' exposure.

Preparation of Washed Mouse Platelets and Platelet Aggregation Studies. Six-week-old FVB mice were injected with PBS containing either 1% normal mouse serum or PEG-rmMGDF (25 µg/kg), a dose known to maximally increase platelet levels (Daw et al., 1998). Five days later, at the time of peak platelet production (Daw et al., 1998), blood was drawn from the abdominal aorta into acid-citrate-dextrose solution (1 volume of acid-citrate-dextrose solution/6 volumes of blood) and pooled (5 ml). Platelet suspensions, washed twice, were prepared as described previously (Hechler et al., 1998a) and the cell count was adjusted to 300 × 10³/µl in the final suspension using an ACT Coulter Diff counter (Beckman-Coulter, Roissy, France). The final suspending medium was Tyrode's buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO₃, 0.3 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 5.5 mM glucose, and 5 mM HEPES, pH 7.3) containing 0.35% human serum albumin and 0.02 U/ml apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were stored at 37°C throughout experiments.

Flow Cytometric Analysis of Lineage-Specific Platelet Surface Markers. Phycoerythrin (PE)-conjugated anti-mouse GPV (DOM2) monoclonal antibody, fluorescein isothiocyanate (FITC)-conjugated anti-GPIIb-IIIa (JON1) and FITC-conjugated anti-GPIbIX (POP1) (Bergmeier et al., 2000) monoclonal antibodies (mAbs) and PE- and FITC-conjugated irrelevant mAbs were kindly provided by Prof. Bernhard Nieswandt (Witten/Herdecke University, Wuppertal, Germany). Washed mouse platelets (1.5 × 10⁶) in 15 µl of Tyrode's buffer containing 0.35% human serum albumin were incubated for 15 min with 0.5 µg of mAb. Samples were then diluted in 500 µl of the same buffer and analyzed on a FACSCalibur fluorescence cytometer (BD Biosciences, San Jose, CA). The fluorescence intensity of 10,000 cells was collected with a logarithmic gain and the binding of mAbs to mouse platelets was represented as the FL1 or FL2 fluorescence intensity relative to that of an irrelevant antibody.

	Results

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PEG-rHuMGDF Increases P2Y₁ Receptor mRNA levels in Y10/L8057 Cells. Northern blot analyses of total RNA from Y10/L8057 cells, using a radiolabeled probe covering the entire translated region of the mouse P2Y₁ gene, indicated the presence of three transcripts with sizes of 7.4, 4.4, and 2.5 kb (Fig. 1A, left). The 4.4-kb transcript seemed to be more abundant than the other two, which were of similar abundance. At 25 ng/ml, a concentration previously shown to maximally promote the differentiation of Y10/L8057 megakaryocytic cells (Zhang et al., 1998; Thompson and Ravid, 1999), PEG-rHuMGDF induced a gradual, time-dependent increase in these three bands (Fig. 1A, left). Levels of the three transcripts seemed to be significantly higher after 8 h stimulation, and we initiated our analyses at this time point because it corresponds to the time of detectable induction of megakaryocyte markers by PEG-rHuMGDF (Thompson and Ravid, 1999) (our equivalent data not shown). After 48-h treatment of Y10/L8057 cells with PEG-rHuMGDF, the three P2Y₁ mRNA bands increased about 3- to 4-fold compared with the corresponding bands in nonstimulated cells (see legend to Fig. 1 for the method of analysis), whereas prolonged incubation with PEG-rHuMGDF indicated that the rise in P2Y₁ mRNA was maximal at this time point (data not shown). Because A2a adenosine receptor mRNA, which is also expressed in Y10/L8057 cells, was not affected by PEG-rHuMGDF (Fig. 1A, right), its enhancement of P2Y₁ mRNA seemed to be selective.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   PEG-rHuMGDF selectively enhances P2Y1 receptor mRNA in Y10/L8057 cells. A, Y10/L8057 cells were cultured in the presence or absence of PEG-rHuMGDF (MGDF) (25 ng/ml) and harvested at the indicated times. Total RNA was extracted and analyzed on Northern blots using 32P-labeled cDNA probes corresponding to the entire translated region of the mouse P2Y1 gene (left) or the A2a adenosine receptor gene (right). Equal loading of RNA (25 µg/lane) was checked with a cDNA probe encoding GAPDH. Levels of P2Y1 mRNA and the fold increase induced by PEG-rHuMGDF were estimated with the electrophoresis documentation and analysis system. Results are the mean ± S.D. from three independent experiments and one representative experiment is shown. B, top, organization of the mouse P2Y1 receptor gene showing the location of the probes used for Northern blot analysis showed bellow. Bottom: Total RNA was extracted from Y10/L8057 cells cultured for 48 h in the presence or absence of PEG-rHuMGDF (25 ng/ml) and analyzed by Northern blotting. 32P-labeled cDNA probes corresponded to the entire translated region (P2Y1 CDS probe), the 5'-untranslated region (5'-UTR probe), and the second exon (exon 2 probe) of the mouse P2Y1 gene.

Stability of P2Y₁ Receptor mRNA in Y10/L8057 Cells. The increase in P2Y₁ receptor mRNA levels during megakaryocyte differentiation in the presence of PEG-rHuMGDF could reflect changes in mRNA stability caused by post-transcriptional modifications and/or changes in the gene transcription rate. Therefore, to better characterize the regulation of P2Y₁ mRNA levels by PEG-rHuMGDF, we assessed the stabilities of P2Y₁ mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in Y10/L8057 cells. Cells cultured for 48 h in the presence or absence of PEG-rHuMGDF (25 ng/ml) were incubated with the transcription inhibitor actinomycin D (5 µg/ml) for up to 10 h, before isolation of total RNA. Northern blot analyses indicated that levels of the three P2Y₁ mRNA transcripts decreased rapidly after addition of actinomycin D (Fig. 2A). Conversely, no decrease in GAPDH mRNA was observed during 10 h incubation with the transcription inhibitor (Fig. 2A). Thus P2Y₁ receptor mRNA levels were normalized at each time point to the corresponding GAPDH mRNA levels. The half-lives of the 7.4-, 4.4-, and 2.5-kb P2Y₁ mRNA transcripts calculated by this method were 1 h, 2.5 h, and 2 h, respectively, and were not modified by PEG-rHuMGDF (Fig. 2B).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   PEG-rHuMGDF does not modify the half-lives of P2Y1 mRNA transcripts. A, Y10/L8057 cells cultured for 48 h in the presence or absence of PEG-rHuMGDF (MGDF) (25 ng/ml) were treated with the transcription inhibitor actinomycin D (5 µg/ml) for up to 10 h. Total RNA was extracted and analyzed on Northern blots using cDNA probes encoding GAPDH or the entire translated region of the mouse P2Y1 gene. B, hybridization bands in the Northern blots of A were quantified with the electrophoresis documentation and analysis system. At each time point, the P2Y1 signals were normalized to the corresponding GAPDH signals and the normalized P2Y1 receptor signals were then expressed as percentages relative to their values at time 0 (100%). One experiment is shown, representative of two experiments yielding identical results.

PEG-rHuMGDF Induces de Novo Synthesis of P2Y₁ Receptor mRNA. To determine whether the increase in steady-state levels of P2Y₁ mRNA resulted from enhanced transcription of the P2Y₁ gene, de novo synthesis of P2Y₁ receptor mRNA was evaluated in nuclear run-on assays, using nuclei isolated from Y10/L8057 cells cultured for 48 h with or without PEG-rHuMGDF (25 ng/ml). Transcription of the P2Y₁ gene increased 2 fold in the presence of PEG-rHuMGDF (Fig. 3). Hence, transcription enhancement was totally responsible for the observed rise in P2Y₁ receptor mRNA levels during PEG-rHuMGDF treatment of Y10/L8057 megakaryocytic cells.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   PEG-rHuMGDF enhances transcription of the P2Y1 gene in Y10/L8057 cells. Nuclear run-on assays were performed on nuclear extracts from Y10/L8057 cells cultured for 48 h in the presence or absence of PEG-rHuMGDF (MGDF) (25 ng/ml), as described under Experimental Procedures. Radioactive newly synthesized mRNA was subsequently hybridized with a plasmid containing the entire translated region of the mouse P2Y1 gene, pUC-19, or cDNA for 18 S ribosomal RNA (10 µg each). Band intensities were quantified with the electrophoresis documentation and analysis system. Data are from one experiment representative of three performed and PEG-rHuMGDF increased the P2Y1 signal 2-fold as assayed by this method.

PEG-rHuMGDF Up-Regulates P2Y₁ Receptor Density on Y10/L8057 Cells. In the absence of any antibody recognizing the mouse P2Y₁ protein, its presence on the surface of Y10/L8057 cells and the effects of PEG-rHuMGDF on the surface density of the receptor were investigated with a radioligand binding assay. The radioligand was [³³P]2MeSADP, an ADP analog previously used to characterize ADP receptors on platelets (Hechler et al., 1998a). [³³P]2MeSADP binding sites corresponding specifically to the P2Y₁ receptor were quantified by measuring binding of the radioligand to Y10/L8057 cells treated or not with PEG-rHuMGDF in the presence or absence of the selective P2Y₁ antagonist A2P5P (1 mM) (Boyer et al., 1996). Nonlinear regression analysis of the data revealed 76,700 ± 7,000 [³³P]2MeSADP binding sites per cell, with an affinity of 73 ± 6 nM, on control cells (Fig. 4A). In the presence of A2P5P, the number of binding sites per cell decreased to 57,800 ± 5,400 and these residual sites displayed an affinity of 80 ± 19 nM. Hence, the "A2P5P-sensitive" sites, corresponding to binding of the radioligand to the P2Y₁ receptor, amounted to 18,900 sites per cell. In the presence of PEG-rHuMGDF (25 ng/ml), [³³P]2MeSADP binding sites increased to 94,100 ± 1,100 sites per cell with an affinity of 64 ± 5 nM. This additional binding corresponded to an increase in the density of P2Y₁ sites because it was displaced by 1 mM A2P5P, whereas the residual "A2P5P-insensitive" binding sites still amounted to 59,200 ± 13,600 sites per cell with an affinity of 81 ± 26 nM. Thus, the P2Y₁ binding sites on Y10/L8057 cells treated with PEG-rHuMGDF represented 34,900 sites per cell and PEG-rHuMGDF enhanced the P2Y₁ receptor density on these cells by a factor of 1.8. 

View larger version (28K): [in this window] [in a new window]   Fig. 4.   PEG-rHuMGDF up-regulates P2Y1 receptor density on Y10/L8057 cells but not in Y10/L8057 cell membranes. Y10/L8057 cells were cultured for 48 h in the presence or absence of PEG-rHuMGDF (MGDF) (25 ng/ml) and P2Y1 receptor densities at the cell surface were evaluated in [33P]2MeSADP binding studies, as described under Experimental Procedures. Saturation experiments in the presence or absence of A2P5P (1 mM), a selective P2Y1 antagonist, were used to distinguish [33P]2MeSADP binding sites corresponding specifically to the P2Y1 receptor. A, incubation of Y10/L8057 cells with PEG-rHuMGDF increased their surface expression of the P2Y1 receptor from 18,900 to 34,900 sites/cell. B, the number of P2Y1 receptor binding sites remained similar in membranes of Y10/L8057 cells cultured in the absence (7.8 ± 0.5 pmol/mg protein) or presence of PEG-rHuMGDF (MGDF) (25 ng/ml) (7.6 ± 1.2 pmol/mg protein). Data were analyzed and plotted with the Ligand program (Rodbard et al., 1986) and the experiment shown is representative of three independent experiments performed in triplicate. Inset, Scatchard plot of the saturation experiments.

In Situ Hybridization Analyses of Megakaryocytes from Mice Injected with PEG-rmMGDF. Our next step was to determine whether PEG-rmMGDF could similarly enhance P2Y₁ gene expression in vivo in primary megakaryocytes. FVB mice were injected with PEG-rmMGDF (50 µg/kg) and levels of P2Y₁ receptor mRNA in spleen megakaryocytes were evaluated by an in situ hybridization technique (see Experimental Procedures). PEG-rmMGDF induces a significant increase in megakaryocyte ploidy and size under these conditions (data not shown; Zimmet et al., 1997). The in situ hybridization method allows one to compare relative levels of gene expression in individual primary megakaryocytes, which are easily recognized because of their size and morphology. P2Y₁ mRNAs were markedly enhanced in megakaryocytes from mice treated with PEG-rmMGDF, compared with control cells treated with vehicle (Fig. 5). Quantitative analyses of silver grains on sections showed averages of 190 ± 24 (n = 11) and 73 ± 12 (n = 11) grains per megakaryocyte in test and control animals, respectively (p < 0.05), which corresponds to a 2.6-fold induction of P2Y₁ receptor mRNA.

View larger version (158K): [in this window] [in a new window]   Fig. 5.   PEG-rmMGDF up-regulates P2Y1 mRNA in vivo in primary megakaryocytes of mice. Spleen sections from FVB mice injected with PEG-rmMGDF (50 µg/kg) or vehicle were hybridized with a 35S-labeled antisense riboprobe for P2Y1 and autoradiography was performed for 4 weeks. Control experiments using the sense riboprobe gave no signal (data not shown). Megakaryocytes were compared on sections from animals treated with PEG-rmMGDF (A) or vehicle (B). Calibration tools allowing the measurement of cell size (OpenLab Scientific Imaging software; Improvision, Lexington, MA) connected to the microscope (Olympus, Melville, NY) were employed in further analyses. However, this method of size analysis provides only a rough estimation, as the cells in these spleen sections are sectioned at different planes in the different samples. Slides were stained with hematoxylin and eosin before examination by bright field microscopy. Magnification, 300×.

Studies of Platelets from Mice Treated with PEG-rmMGDF. Circulating platelet levels rose from 823 × 10³ ± 125 × 10³ (n = 17) to 1,600 × 10³ ± 163 × 10³ platelets/µl (n = 9) after administration of PEG-rmMGDF (25 µg/kg) to mice. Platelet P2Y₁ receptor densities were determined by measuring the binding of [³³P]2MeSADP to washed platelets from mice injected or not with PEG-rmMGDF (25 µg/kg), in the presence or absence of A2P5P (1 mM) (Hechler et al., 1998a). Saturation experiments revealed 1,075 ± 9 [³³P]2MeSADP binding sites per platelet, with an affinity of 11 ± 1 nM, on control platelets (Fig. 6A). In the presence of A2P5P, the number of binding sites per platelet decreased to 742 ± 23 and these residual sites displayed an affinity of 9 ± 1 nM. Hence, the P2Y₁ receptor density was calculated to be 333 receptors per platelet in control mice. Similarly, in mice receiving PEG-rmMGDF, [³³P]2MeSADP binding sites represented 975 ± 54 sites per platelet with an affinity of 9 ± 1 nM (Fig. 6A). Addition of A2P5P reduced [³³P]2MeSADP binding sites to 627 ± 72 sites per platelet with an affinity of 8 ± 1 nM (Fig. 6A), giving a calculated P2Y₁ receptor density of 348 receptors per platelet. Thus, injection of PEG-rmMGDF caused no significant change in platelet P2Y₁ receptor density in mice.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   PEG-rmMGDF does not alter platelet P2Y1 receptor density or reactivity in mice. A, saturation binding of [33P]2MeSADP to platelets from mice treated or not with PEG-rmMGDF (25 µg/kg), in the presence or absence of A2P5P (1 mM). Points are the means of triplicate determinations in a single experiment representative of two independent experiments. Inset, Scatchard plot. B, in vitro aggregation profiles of platelets from mice treated or not with PEG-rmMGDF (25 µg/kg). Aggregation in response to ADP (5 µM or 1 µM) was similar in control animals and mice receiving PEG-rmMGDF (top and middle). In the presence of the P2Y12 antagonist AR-C69931 MX (10 µM), ADP (5 µM) induced identical aggregation of platelets from mice injected with PEG-rmMGDF and control cells (bottom).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Flow cytometric determination of lineage-specific markers on murine platelets. GPIIbIIIa, GPV, and GPIbIX expression on platelets from control mice (white bars) and mice injected with PEG-rmMGDF (25 µg/kg) (black bars) was detected with the JON1, DOM2, and POP1 mAbs, respectively. Flow cytometric data are expressed as the fluorescence intensity of the PE- (JON1) or FITC-conjugated mAb (DOM2 and POP1) and mean values were calculated from two separate experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

To determine whether Mpl ligand could affect expression of the P2Y₁ receptor during megakaryocytopoiesis, we performed studies in the mouse megakaryocytic cell line Y10/L8057. These cells can be generated in large quantities and have been shown to respond to a truncated form of Mpl ligand, PEG-rHuMGDF, through an increase in ploidy and enhanced expression of platelet specific proteins such as GPIIb (2- to 3-fold) and platelet factor 4 (6 fold) (Zhang et al., 1998; Thompson and Ravid, 1999), thus acquiring the characteristics of differentiated megakaryocytes. The in vivo effects of Mpl ligand on levels of P2Y₁ mRNA in primary megakaryocytes were also examined in mice.

In accordance with previous reports describing RT-PCR amplification of P2Y₁ cDNA from various human megakaryoblastic cell lines (Léon et al., 1997; Jin et al., 1998b; Kaushansky, 1999), P2Y₁ mRNA was found to be present in Y10/L8057 cells (Fig. 1). Because the phenotype of control untreated Y10/L8057 cells is megakaryoblastic, with a low percentage of acetylcholinesterase-positive cells and a ploidy distribution displaying a majority of 2N cells (Zhang et al., 1998), the P2Y₁ receptor seems to be expressed early in megakaryocyte maturation. Three different P2Y₁ mRNAs with sizes of 2.5 kb, 4.4 kb, and 7.4 kb were present in Y10/L8057 cells. The 4.4-kb P2Y₁ mRNA was the most abundant in resting cells, whereas the 7.4- and 2.2-kb P2Y₁ mRNAs were of similar abundance (Fig. 1). Northern blot analyses using probes corresponding to different regions of the mouse P2Y₁ gene indicated that the different sizes could result from alternative splicing of the unique intron, together with use of various polyadenylation sites present in the gene. The production of P2Y₁ mRNA of three different sizes might play a role in modulating translation efficiency and therefore receptor expression levels. These three transcripts might also be involved in the tissue-specific distribution of the receptor. It should be pointed out, moreover, that the occurrence of three P2Y₁ transcripts is not a property of Y10/L8057 cells; three molecules were also detected by Northern blotting in mouse heart, brain, liver and kidney (C. Léon, personal communication).

Although the half-lives of the three P2Y₁ mRNAs were not affected by PEG-rHuMGDF (Fig. 2), the very short lives of these transcripts (1 to 2.5 h) suggests that expression of the P2Y₁ receptor could be rapidly modulated by changes in gene transcription or mRNA stability. Moreover, because platelets are unable to transcribe new mRNA because they have no nuclei, young platelets may be able to synthesize P2Y₁ receptor protein from reminiscent megakaryocyte mRNA, whereas old platelets may not. Despite the current lack of experimental data concerning the stability of the platelet P2Y₁ protein, this means that the short half-life of P2Y₁ mRNA could be of functional importance in terms of platelet reactivity.

In the present study, we demonstrated that the increase in P2Y₁ mRNA levels in Y10/L8057 cells arises through a direct action of PEG-rHuMGDF on the rate of transcription of the P2Y₁ gene (Fig. 3), which implies that the gene contains specific elements responsive to PEG-rHuMGDF. It was recently shown that several transcription factors including Sp1 (Zhang et al., 1998), PU.1/Spi-1 (Doubeikovski et al., 1997), and CREB (Zauli et al., 1997) are activated by Mpl ligand and are responsible for the cytokine-dependent transcriptional activation of genes, such as those of GPIIb (Doubeikovski et al., 1997; Zauli et al., 1997) and cyclin D3 (Zhang et al., 1998). It remains to be determined whether one or more of these transcription factors is responsible for the enhancement by PEG-rHuMGDF of transcription of the P2Y₁ gene.

The rise in P2Y₁ receptor mRNA correlates with an increase in P2Y₁ protein levels at the surface of Y10/L8057 cells (Fig. 4A). This increase in P2Y₁ mRNA induced by PEG-rHuMGDF is not unique to a megakaryocytic cell line; it also occurred in the primary megakaryocytes of mice injected with the recombinant murine cytokine (Fig. 5). Although circulating platelet levels rose 2-fold in these mice, the platelet P2Y₁ receptor density (Fig. 6A) and aggregation profile in response to ADP (Fig. 6B) were not significantly modified by PEG-rmMGDF treatment. Thus, the enhanced P2Y₁ expression in megakaryocytes led to an increase in neither the density of the P2Y₁ receptor on platelets nor their reactivity to ADP. It is important to note that PEG-rHuMGDF did not increase the density of P2Y₁ receptors per milligram of protein of Y10/L8057 cell membranes. Thus, it seems that the rise in P2Y₁ receptor expression induced by Mpl ligand parallels the well documented global increase in megakaryocyte size and ploidy, the extensive development of demarcation membranes that precedes platelet production, and the rise in platelet levels (Kaushansky, 1999), so that the P2Y₁ receptor density on the resultant platelets remains stable. A similar phenomenon has been reported for GPIIb, which is enhanced in megakaryocytes under Mpl ligand treatment (Zauli et al., 1997), without any corresponding increase in levels of the protein in platelets (Harker et al., 1996b; O'Malley et al., 1996). Furthermore, expression of the platelet surface glycoproteins GPIIbIIIa, GPIbIX, and GPV remained comparable in the present work in mice treated with PEG-rmMGDF and control animals (Fig. 7).

Mpl ligand is currently under clinical investigation as a drug to stimulate platelet production in patients with bone marrow failure. Plasma levels of Mpl ligand are inversely related to the platelet count and it has also been clearly established in vitro that this cytokine potentiates platelet aggregation in response to various agonists including ADP (Oda et al., 1996, 1999). If this were applicable in vivo, a cumulative effect would have been expected to create a risk of thrombotic complications in patients receiving Mpl ligand. However, a recent study reported no change in the ADP response of washed platelets isolated from healthy human volunteers treated with recombinant Mpl ligand (Harker et al., 2000), in agreement with an earlier finding in nonhuman primates (Harker et al., 1996b) and with our current findings in mice. Because the platelet P2Y₁ receptor plays a key role in hemostasis and thrombosis (Cattaneo and Gachet, 1999; Gachet, 2001), this apparent lack of increased platelet reactivity to ADP suggests that clinical use of Mpl ligand to promote platelet production might not in fact be associated with adverse effects of platelet hyper-reactivity.