Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Authors

  • B. Hechler,

    1. INSERM U.311, Etablissement Français du Sang-Alsace, Strasbourg Cedex, France
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    • 1

      Present address: INSERM U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, B.P. N°36, 67065 Strasbourg Cedex, France.

  • Y. Zhang,

    1. Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA; and
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  • A. Eckly,

    1. Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA; and
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  • J-P. Cazenave,

    1. Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA; and
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  • C. Gachet,

    1. Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA; and
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  • K. Ravid

    1. INSERM U.311, Etablissement Français du Sang-Alsace, Strasbourg Cedex, France
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Dr Katya Ravid, Department of Biochemistry, K225, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA. Tel.: + 1 617 6385053, fax: + 1 617 6385054, e-mail: ravid@med-biochem.bu.edu

Abstract

Summary.  In order to investigate the role of the platelet P2Y1 receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [33P]2MeSADP in the presence or absence of the selective P2Y1 antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y1 receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y1 receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5′-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y1 receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y1 expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y1 receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y1 receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.

Introduction

Adenosine 5′-diphosphate (ADP), one of the main platelet activators, plays a central role in normal hemostasis and in the pathogenesis of arterial thrombosis [1]. Although by itself in vitro only a weak platelet aggregating agent, ADP is a necessary cofactor for normal activation of platelets by other agonists such as thrombin or collagen, which induce its secretion from the so-called dense granules where it is stored at near molar concentrations [1]. Coactivation of two G protein-coupled receptors, P2Y1 and P2Y12, is necessary for normal platelet aggregation in response to ADP. The recently identified P2Y12 receptor [2–4], coupled to inhibition of adenylyl cyclase through a Gi2 protein, is responsible for amplification of the platelet aggregation induced by ADP [5,6] and other aggregating agents [1]. The role of this receptor in thrombosis is well established as it is the target of the anti-thrombotic drug clopidogrel (Plavix®) [1,7], which is used clinically for the secondary prevention of thrombosis in patients with established vascular disease [8]. The P2Y1 receptor, a Gαq protein-coupled receptor, is responsible for the mobilization of intracellular calcium stores. Pharmacological studies using selective P2Y1 antagonists [9–11] and investigations in P2Y1 knockout mice [12,13] have identified this receptor as necessary for ADP-induced platelet shape change and the initiation of aggregation. Studies in P2Y1 knockout mice also pointed to its essential role in experimental thrombosis [12–14].

As compared with strong platelet activators like thrombin, collagen or thromboxane A2 (TXA2), ADP induces only weak and reversible platelet aggregation without release of the granule contents [15,16]. At the intracellular level, ADP triggers weak P2Y1 receptor-mediated activation of PLCβ, leading to the production of small amounts of inositol 1,4,5-trisphosphate (IP3) and a moderate increase in intracellular calcium levels as compared with thrombin or TXA2. The density of P2Y1 receptors on the platelet surface is relatively low (130 receptors per platelet) [17], in comparison for example to the density of TXA2 receptors (1500 receptors per platelet) [18]. Whether the globally weak platelet activation induced by ADP could be due to the low density of the platelet P2Y1 receptor density has never been investigated.

One way to address these points is to selectively overexpress the platelet P2Y1 receptor gene in mice. Such a model should, first, allow evaluation of the consequences of P2Y1 receptor overexpression in terms of platelet reactivity and sensitivity of the mice to thrombosis. Second, it should permit one to determine whether the weak platelet activation induced by ADP is due to the low cell surface density of P2Y1 receptors or to activation of a specific transduction pathway. In this objective, transgenic (TG) mice with targeted overexpression of the P2Y1 receptor selectively in the megakaryocytic lineage were generated using the promoter of the rat platelet factor 4 (PF4) gene, which has previously been shown to direct transgene expression solely to the megakaryocyte/platelet lineage [19].

Platelets of TG mice overexpressing the P2Y1 receptor displayed hyper-reactivity in vitro, as reflected by increased aggregation and induction of the release reaction in response to ADP. The hyper-reactivity of these platelets was also apparent in a model of in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. These results highlight the role of the platelet P2Y1 receptor in hemostasis and thrombosis and hence reinforce the concept of this receptor as a potential target for new anti-thrombotic drugs. Furthermore, they imply that controlled expression of the P2Y1 receptor gene is important for normal platelet activation, suggesting that it would be of interest to study the regulation of the expression of this gene.

Materials and methods

Chemicals

2-Methylthioadenosine-5′-diphosphate (2MeSADP) was from Research Biochemicals Incorporated (Natick, MA, USA) and ADP from Sigma (Saint Louis, MO, USA, or Saint Quentin-Fallavier, France). Prostaglandin E1 (PGE1), type I bovine collagen, U46619, MRS2179 (2′-deoxy-N6-methyladenosine-3′,5′-bisphosphoric acid) and essentially fatty acid-free human serum albumin were from Sigma (Saint Quentin-Fallavier, France). Aspirin was purchased from Synthelabo (Le Plessis Robinson, France). Human fibrinogen was from Kabi (Stockholm, Sweden), fura-2/acetoxymethyl ester (fura-2/AM) from Calbiochem (Meudon, France) and [3H]serotonin ([3H]5-HT) and the cyclic adenosine-3′,5′-monophosphate (cAMP) assay kit from Amersham (Les Ulis, France). Apyrase was purified from potatoes as previously described [20]. [33P]2MeSADP was provided by Du Pont NEN (Le Blanc Mesnil, France) and AR-C69931MX was from Astra Charnwood (Loughborough, UK). The anesthetic drugs xylazine (Rompun) and ketamine (Imalgene 1000) were from Bayer (Paris, France) and Merial (Lyon, France), respectively.

Plasmid construction

The plasmid PF4-SV40, a pUC-based plasmid containing 1.1 kb of the rat PF4 promoter followed by the simian virus 40 (SV40) small-T intron and polyadenylylation sequences, was cut at the NcoI and SalI unique sites. The SV40 splicing and polyadenylylation sites are known to allow enhanced expression of transgenes in the mouse [21]. The 1.3 kb coding sequence of the mouse P2Y1 gene (GenBank accession number AJ245636, gift from C. Léon, INSERM U.311, France) was excised from the pcDNA3 vector with KpnI and XhoI enzymes. This region extends from 129 bp upstream of the translation start to 116 bp downstream of the stop codon. XhoI and SalI generate cohesive ends and ligation between the PF4-SV40 plasmid and the P2Y1 coding sequence was performed in the presence of the NcoI/KpnI linker 5′-CAT GGT AC-3′. The resulting plasmid, referred to as PF4-P2Y1-SV40, was purified on a cesium chloride gradient, confirmed by DNA sequencing and subsequently digested with NdeI to release the vector sequences. The 3-kb vector-free DNA fragment was then purified on a 0.8% agarose gel with a Geneclean kit (Bio101, Vista, CA, USA) and used for the production of TG mice as described below.

Generation of transgenic mice

DNA was microinjected into male pronuclei of one-cell FVB/N strain (Taconic, Germantown, NY, USA) mouse embryos to produce transgenic mice [19]. Genomic DNA was isolated from the tails of potential founder mice, digested with BanII and tested for transgene integration by Southern blotting, using a BanII-BanII fragment corresponding to the coding sequence of the P2Y1 gene as probe. Transgene expression was detected by RT-PCR followed by Southern blotting with an SV40 probe. Briefly, total RNA was isolated from the bone marrow of TG or wild-type (WT) mice with Trizol reagent (Gibco BRL, Gaithersburg, MD, USA), as described previously [22,23]. First-strand cDNA synthesis was carried out according to the protocol of a SMARTTM cDNA Library Construction kit (Clontech, Palo Alto, CA, USA), using specific sense 5′-GCA GTG GTA ACA ACG CAG AGT ACG CGG G-3′ and antisense 5′-GCA GTG GTA ACA ACG CAG AGT ACT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTV N-3′ primers. After cDNA amplification in an ExpandTM Long Template PCR system (Boehringer, Mannheim, Germany), using the same specific sense primer and 20 cycles of PCR, the product was analyzed on Southern blots with the SV40 probe.

Bleeding time measurement

The bleeding time was measured after severing a 3-mm segment of tail of a 6- to 10-week-old WT or TG mouse. The amputated tail was immersed in 0.9% isotonic saline at 37 °C, and the time required for the stream of blood to stop was defined as the bleeding time. If no cessation of bleeding occurred after 10 min, the tail was cauterized and 600 s was recorded as the bleeding time.

Preparation of washed mouse platelets and platelet aggregation studies

Blood was drawn from the abdominal aorta into acid/citrate/dextrose (ACD) anticoagulant (1 volume ACD/6 volumes blood), pooled (5 mL), and twice-washed platelet suspensions were prepared as described previously [12]. The final resuspending medium was Tyrode's buffer (137 mmol L−1 NaCl, 2 mmol L−1 KCl, 12 mmol L−1 NaHCO3, 0.3 mmol L−1 NaH2PO4, 2 mmol L−1 CaCl2, 1 mmol L−1 MgCl2, 5.5 mmol L−1 glucose, 5 mmol L−1 HEPES, pH 7.3) containing 0.35% human serum albumin and 0.02 U mL−1 apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were stored at 37 °C throughout experiments and cell counts were adjusted to 200 × 103 µL−1 in the final suspension using an ACT Coulter DiffTM counter (Coulter-Beckmann, Roissy, France). In some experiments, platelets were treated with 1 mmol L−1 aspirin for 15 min in the second wash before final resuspension in Tyrode's buffer containing albumin and apyrase.

Aggregation was measured at 37 °C by a turbidimetric method in a dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). A 450-µL aliquot of platelet suspension was stirred at 1100 r.p.m. and activated by addition of 50 µL of the appropriate agonist, fibrinogen (0.2 mg mL−1) being added before stimulation with ADP. The extent of aggregation was estimated quantitatively by measuring the maximum curve height above baseline.

Binding of [33P]2MeSADP to washed mouse platelets

Binding of [33P]2MeSADP to washed mouse platelets was determined as previously described [6] by incubation of [33P]2MeSADP (0.5 nmol L−1; 300 000 disintegrations per minute) with washed platelet suspensions (3 × 105 platelets µL−1) for 5 min at 37 °C in a final volume of 200 µL. Experiments were started by addition of platelets to the reaction mixture and were carried out in triplicate and in the presence or absence of 10 µmol L−1 MRS2179, a potent P2Y1 antagonist [24]. The reaction was terminated by dilution in 4 mL of cold buffer (Tyrode's buffer containing no Ca2+ or Mg2+) and rapid filtration through Whatman GF/C glass fiber filters under vacuum, after which the tubes and filters were rinsed four times with 2 mL of ice-cold buffer. Radioactivity bound to platelets on the filters was measured by scintillation counting (Wallac 1409 counter, Turku, Finland). Non-specific binding, determined by similar incubation of platelets with 1 µmol L−1 unlabeled 2MeSADP, amounted to about 5% of the total binding. Saturation experiments were performed using a single concentration of [33P]2MeSADP in the presence of increasing concentrations of unlabeled 2MeSADP and data were analyzed and plotted with the ligand program [25].

[Ca2+]i measurements

Mouse platelets resuspended in the second washing solution (about 600 × 103 platelets µL−1 in Tyrode's buffer without calcium) were loaded with 15 µmol L−1 fura-2/AM for 45 min at 37 °C in the dark [12,26]. After centrifugation, the platelet pellet was resuspended in the same buffer (200 × 103 platelets µL−1) containing 0.02 U mL−1 apyrase and 0.1% essentially fatty acid-free human serum albumin and kept at room temperature throughout experiments. Aliquots of fura-2/AM-loaded platelets were transferred to a 10 × 10 mm quartz cuvette maintained at 37 °C and fluorescence measurements were performed under continuous stirring in a PTI Deltascan spectrofluorimeter (Photon Technology International Inc, Princetown, NJ, USA). The excitation wavelength was alternately fixed at 340 or 380 nm and fluorescence emission was determined at 510 nm.

Measurement of cAMP levels

A 450-µL aliquot of washed mouse platelets was stirred at 1100 r.p.m. in an aggregometer cuvette and stimulated by addition of 10 µmol L−1 PGE1 followed after 30 s by 1 µmol L−1 ADP or vehicle (Tyrode's buffer containing no Ca2+ or Mg2+). One minute later, the reaction was stopped by adding 50 µL of ice-cold 6.6 mol L−1 perchloric acid and cAMP was extracted as previously described [26] and quantified with a commercial radioimmunoassay kit.

Platelet secretion

Platelets resuspended in the first washing buffer (600 × 103 platelets µL−1) were labeled for 15 min with 5-hydroxy[3H]tryptamine ([3H]5-HT) (1 µCi mL−1) at 37 °C and washed as described above. A 450-µL aliquot of washed platelets was stirred at 1100 r.p.m. in an aggregometer cuvette maintained at 37 °C. Platelets were activated by addition of 50 µL of various agonists for 3 min, the reaction was stopped by adding 100 µL of 10% formaldehyde and samples were centrifuged at 10 000 × g for 5 min, after which the radioactivity in the supernatants was measured by liquid-scintillation counting (Wallac 1409 counter, Turku, Finland). The platelet dense granule secretion was determined by calculating the release of [3H]5-HT and expressed as the percentage of the total [3H]5-HT content.

Electron microscopy

A 450-µL aliquot of platelet suspension from either WT or TG mice was fixed in the aggregometer cuvette by addition of an equal volume of fixative solution (2.5% glutaraldehyde in 0.1 mol L−1 sodium cacodylate buffer containing 2% sucrose, 305 mOsm, pH 7.3) previously warmed to 37 °C. After 5 min at 37 °C, the platelets were centrifuged at 12 000 × g for 20 s and the pellet was resuspended in fixative solution for 45 min. The cells were then rinsed, postfixed for 1 h at 4 °C with 1% osmium tetroxide in cacodylate buffer, washed in the same buffer, dehydrated in graded ethanol solutions and embedded in epon. The resin was allowed to polymerize at 50 °C for 2 days. Ultrathin sections (100 nm) were stained with lead citrate and uranyl acetate and examined under a Philips CM 120 BioTwin (Eindhoven, the Netherlands) transmission electron microscope (120 kV).

In vivo platelet aggregation

Six- to 10-week-old male mice weighing 20–30 g were anesthetized and the jugular vein was exposed surgically. A mixture of collagen (0.125 mg kg−1) and epinephrine (60 µg kg−1) was injected within an infusion time frame of 3–4 s as previously described [12,27]. Two minutes later, blood was drawn from the abdominal aorta into EDTA anticoagulant (6 mmol L−1) and platelets were counted in an ACT Coulter DiffTM counter (Coulter-Beckmann, Roissy, France).

Bone marrow preparation and acetylcholine esterase assay

Bone marrow was harvested from the femurs of transgenic mice [28] and megakaryocytes were identified by in situ staining for acetylcholine esterase [29].

Results

Generation of transgenic mice overexpressing the P2Y1 receptor in platelets

The construct used to generate TG mice, referred to as PF4-P2Y1-SV40, contains 1104 base pairs of the 5′ upstream region of the rat PF4 gene (promoter region), which has been shown to direct transgene expression solely to the megakaryocyte/platelet lineage [28]. This promoter region was linked to the coding sequence of the mouse P2Y1 receptor gene tagged at its 3′ end with the SV40 small-T intron and polyadenylation sequences (Fig. 1a). Among the three transgenic lines produced, one expressed the transgene. Southern blot analyses of genomic DNA revealed that this transgenic founder had integrated approximately 15 copies of the transgene, based on a comparison with the intensity of the endogenous P2Y1 gene (Fig. 1b). Digestion with different restriction enzymes indicated that the integrated transgene was not rearranged (data not shown). Offspring of this mouse inherited the transgene in a mendelian fashion. Its expression was confirmed by analyzing RNA isolated from total bone marrow cells by RT-PCR coupled to Southern blotting, using a probe specific for the SV40 tag of the transgene. The results showed that the bone marrow cells of the offspring of this mouse expressed the transgene (Fig. 1c). All subsequent investigations were performed on mice that were homozygous for the transgene as confirmed by breeding experiments. Non-transgenic littermates were used as controls.

Figure 1.

Generation of transgenic (TG) mice overexpressing a PF4-driven P2Y1 receptor gene. (a) Schematic representation of the construct PF4-P2Y1-SV40 used to generate TG mice. The coding sequence of the mouse P2Y1 receptor gene was subcloned into a pUC-based vector containing the rat PF4 promoter and the simian virus 40 (SV40) small-T intron and polyadenylation sequences. NdeI restriction sites were used to free the plasmid from the vector sequences prior to microinjection into one-cell mouse embryos. (b) Identification of TG mice. One example of a Southern blot analysis of tail genomic DNA from a founder line. DNA digested with BanII and the BanII fragment of the construct shown in (a) was used as a probe. (c) Transgene expression in TG mice. Transgene expression was followed by reverse transcription (RT) of RNA prepared from the bone marrow of WT mice or of F1 progeny of TG mice, followed by PCR amplification and Southern blot analysis of the PCR products using a 0.5-kb SV40 DNA fragment as a probe (see Methods). The figure focuses on the TG line which expressed the transgene. The RT reaction was carried out in the absence [control (–) or presence (+) of reverse transcriptase]. (d) Platelet P2Y1 receptor levels in TG and control mice. Saturation binding of [33P]2MeSADP to WT or TG mouse platelets was measured in the presence or absence of 10 µmol L−1 MRS2179. Data are from a single experiment representative of three experiments performed and each point is the mean of triplicate determinations. Insert: Scatchard plot.

The density of the P2Y1 receptor on the platelet surface was determined by binding studies using the ADP analog [33P]2MeSADP, previously employed to characterize ADP receptors on platelets [30]. Binding sites corresponding specifically to the P2Y1 receptor were evaluated by measuring the binding of [33P]2MeSADP to WT or TG mouse platelets in the presence or absence of the selective P2Y1 antagonist MRS2179 [17,24]. The total number of [33P]2MeSADP binding sites per platelet increased from 693 ± 29 (n = 3) in WT to 802 ± 54 (n = 3) in TG platelets (Fig. 1d). This increase corresponded to a specific rise in P2Y1 receptor density, as in the presence of MRS2179 (10 µmol L−1) the residual number of binding sites per platelet, corresponding to the P2Y12 receptor, was identical in WT (543 ± 9, n= 3) and TG (527 ± 20, n= 3) cells (Fig. 1d). Thus, WT mouse platelets bore 150 ± 31 (n = 3) P2Y1 receptors and TG mouse platelets 276 ± 34 (n = 3), which represents an 84% increase in P2Y1 receptor density.

The general health and life span of the homozygous TG mice appeared to be normal. Platelet counts lay in the normal range (Table 1) and transmission electron microscopy revealed no abnormalities of platelet morphology (data not shown). The number of megakaryocytes in the bone marrow was also similar between WT and TG mice (Table 1), ruling out a significant role of the P2Y1 receptor in platelet production.

Table 1.  Platelet count ( µL−1 of blood) and megakaryocyte count (megakaryocyte per 5 × 105 bone marrow cells) of WT and TG mice
Platelet count (platelets µL−1 blood)1156 000 ± 87 8801257 000 ± 52 620(Not significant)
Megakaryocyte number (5 × 105 bone marrow cells)80.1 ± 9.991.5 ± 13.7(Not significant)

TG mice display a shortened bleeding time

The bleeding time, which reflects in vivo primary hemostasis, was shortened significantly in TG mice (87 ± 4 s, n = 21) as compared with WT mice (167 ± 33 s, n = 19, P = 0.0170) (Fig. 2), suggesting a repercussion of P2Y1 receptor overexpression on platelet reactivity.

Figure 2.

Bleeding time of WT and TG mice. Each point represents one individual (WT, n= 19; TG, n= 21) and the small bars denote averages. The bleeding time was significantly although mildly shortened in TG mice (87 ± 4 s, n = 21) as compared with WT mice (167 ± 33 s, n = 19) (*P = 0.0170).

Hyperaggregatory responses of TG mouse platelets

Functional studies showed that aggregation in response to ADP (1 µmol L−1) was strongly increased in TG (56 ± 3%) as compared with WT mouse platelets (35 ± 2%, n = 7, P < 0.0001) (Fig. 3a). Interestingly, the extent of ADP-induced platelet aggregation in heterozygous mice displaying 50% expression of the transgene was 43 ± 3% as compared with 31 ± 2% (n = 4, P = 0.0176) in their WT counterparts (data not shown). This would indicate a causal link between the P2Y1 receptor gene dosage and the amplitude of ADP-induced platelet aggregation.

Figure 3.

In vitro platelet aggregation studies. (a) Aggregation profile of WT or TG mouse platelets stimulated with 1 µmol L−1 ADP. Tracings are from one experiment representative of seven independent experiments yielding identical results. (b) Dose–response curves for ADP-induced aggregation of TG and WT mouse platelets in the presence of 2 µmol L−1 AR-C69931MX, a selective P2Y12 antagonist. Curves represent the mean of four independent experiments and bars show the SEM. (c) Aggregation profile of WT or TG mouse platelets in response to 1.25 µg mL−1 collagen (left panel) or 2.5 µg mL−1 collagen (right panel). Tracings are from one experiment representative of three independent experiments yielding identical results.

In order to focus solely on the role of the P2Y1 receptor, ADP-induced platelet aggregation was evaluated in the presence of the selective P2Y12 receptor antagonist AR-C69931MX (2 µmol L−1) [31]. Aggregation in response to ADP (0.3 µmol L−1 to 10 µmol L−1) was increased in TG as compared with WT mouse platelets under these conditions (Fig. 3b). The maximal aggregation induced by 10 µmol L−1 ADP was significantly increased in TG (25 ± 5%) relative to WT platelets (9 ± 3%, n = 4, P = 0.0442) (Fig. 3b), while the 50% efficacy concentration (EC50) of ADP-induced platelet aggregation was two times lower in TG mouse platelets (1.6 ± 0.3 µmol L−1) than in the WT cells (3.4 ± 0.8 µmol L−1).

The platelet aggregation induced by a low concentration of collagen (1.25 µg mL−1) was also enhanced in TG as compared with WT mouse platelets (Fig. 3c, left panel), but this effect disappeared at a higher collagen concentration (2.5 µg mL−1) (Fig. 3c, right panel). These results confirm the previously proposed role of the P2Y1 receptor in the early phases of collagen-induced platelet activation [12]. Aggregation in response to thrombin (0.1 U mL−1) or the TXA2 analog U46619 (2 µmol L−1) was similar in WT and TG mouse platelets (data not shown).

Effects of platelet P2Y1 receptor overexpression on ADP-induced signal transduction pathways

The P2Y1-mediated mobilization of intracellular calcium stores triggered by a low concentration of ADP (0.1 µmol L−1) was increased in TG as compared with WT mouse platelets (Fig. 4a, left panel). Quantitatively, the ADP-induced rise in [Ca2+]i above basal levels was 120 ± 3 nmol L−1 in WT and 216 ± 2 nmol L−1 (n = 3, P < 0.0001) in TG platelets. At a higher concentration of ADP (10 µmol L−1), although the magnitude of the [Ca2+]i rise was not significantly different between WT (322 ± 29 nmol L−1) and TG (358 ± 31 nmol L−1) (n = 3) mouse platelets, the signal was more sustained and less reversible in TG platelets than in the WT cells (Fig. 4a, right panel). Thus, 2 min after platelet stimulation, the intracellular calcium rise was 231 ± 16 nmol L−1 in TG but only 118 ± 2 nmol L−1 (n = 3, P= 0.0023) in WT mouse platelets.

Figure 4.

[Ca2+]i and cyclic AMP measurements. (a) [Ca2+]i levels in WT or TG mouse platelets stimulated with 0.1 µmol L−1 ADP (left panel) or 10 µmol L−1 ADP (right panel). The data are presented as arbitrary units (a.u.) of fluorescence and are from one experiment representative of three independent experiments giving identical results. (b) Cyclic AMP levels in WT (left panel) and TG (right panel) mouse platelets activated with 10 µmol L−1 PGE1 followed by 1 µmol L−1 ADP. Data are mean values (± SEM) from three separate experiments, each performed in triplicate.

Concerning the inhibition of adenylyl cyclase activity mediated by P2Y12 receptor activation, ADP (1 µmol L−1) reduced cyclic AMP levels to a similar extent in PGE1 (10 µmol L−1)-stimulated WT and TG platelets (Fig. 4b). This suggests that overexpression of the P2Y1 receptor does not interfere with the transduction pathways associated with activation of the P2Y12 receptor.

Overexpression of the platelet P2Y1 receptor allows ADP to induce the release reaction

In the presence of physiological concentrations of external calcium, ADP is a weak platelet activator inducing reversible aggregation without release of the granule contents [15,16]. Consistent with these observations, in WT mouse platelets loaded with [3H]serotonin ([3H]5-HT), ADP (1–100 µmol L−1) induced secretion of less than 8% of the serotonin content (Fig. 5a, upper panel). In contrast, stimulation of TG mouse platelets with ADP (1–100 µmol L−1) led to a dose-dependent increase in [3H]5-HT secretion, which reached 24 ± 3% in response to 100 µmol L−1 ADP as compared with 8 ± 1% in the WT cells (Fig. 5a, upper panel) (n = 6, P = 0.0004). The ADP analog 2MeSADP (100 µmol L−1), the most potent P2Y1 receptor agonist [32], induced a serotonin release of 34 ± 3% in TG platelets but of only 11 ± 1% in WT mouse platelets (n = 4, P = 0.0006) (Fig. 5a, upper panel). Arachidonic acid (100 µmol L−1), which is taken up by platelets and causes their aggregation through its conversion to TXA2, elicited on the other hand similar [3H]5-HT release from WT (21 ± 4%) and TG (19 ± 3%) platelets (data not shown) (n = 5, P= 0.7223). ADP (100 µmol L−1)-induced secretion of granule contents in TG mouse platelets was inhibited by aspirin (1 mmol L−1), indicating that it was dependent on synthesis of TXA2 (Fig. 5a, lower panel).

Figure 5.

ADP induces the release reaction of TG mouse platelets. (a) Upper panel: [3H]5-HT release from prelabeled WT or TG mouse platelets in response to increasing concentrations of ADP, mean values (± SEM) from six independent experiments. 2MeSADP (100 µmol L−1), the most potent P2Y1 receptor agonist, was also employed, as indicated, and results are mean values (± SEM) from four separate experiments. Lower panel: ADP (100 µmol L−1)-induced release reaction of TG mouse platelets was inhibited by aspirin (1 mmol L−1). (b) Transmission electron micrographs of platelet aggregates induced by ADP (100 µmol L−1) for 3 min of WT (left panel) or TG mice (right panel). Degranulated cells were observed in the center of the aggregates of TG platelets (right panel) (see arrow) but not in aggregates of WT platelets (left panel).

The selective release of the granule contents triggered by ADP (100 µmol L−1) in TG mouse platelets was confirmed by transmission electron microscopy, where degranulated cells were observed in the center of the aggregates (Fig. 5b, right panel) but not in aggregates of WT platelets (Fig. 5b, left panel). Furthermore, these images show that ADP (100 µmol L−1) induced secretion of the alpha granule contents only in TG mouse platelets (Fig. 5b).

Enhanced sensitivity of TG mice overexpressing the P2Y1 receptor to in vivo induced platelet aggregation

As platelets from mice overexpressing the P2Y1 receptor displayed increased sensitivity to aggregating agents in vitro, it was of interest to determine whether this might have an impact in vivo. Hence, experiments were performed in a model of in vivo platelet aggregation induced by infusion of a mixture of collagen (0.125 mg kg−1) and epinephrine (0.6 µg kg−1) into the jugular vein [27,33]. Two minutes after injection of the agonists, blood was collected from the abdominal aorta and platelets were counted. As noted above, platelet counts before injection were comparable in WT and TG mice (Fig. 6). The chosen concentrations of collagen and epinephrine caused no significant decrease in the platelet count of WT mice (1369 × 103 ± 48 × 103 platelets µL−1) as compared with saline injection (1353 × 103 ± 29 × 103 platelets µL−1) (n = 6, P = 0.7857) (Fig. 6). Conversely, the same mixture induced a significant decrease in the platelet count of TG mice (857 × 103 ± 80 × 103 platelets µL−1) as compared with saline injection (1332 × 103 ± 36 × 103 platelets µL−1) (n = 7, P < 0.0001). Mice overexpressing the P2Y1 receptor selectively in the platelet lineage thus appeared to be more sensitive to in vivo platelet aggregation triggered by collagen and epinephrine than WT mice.

Figure 6.

Platelet counts in WT mice and TG mice overexpressing the P2Y1 receptor after injection of either saline or a mixture of collagen (0.125 mg kg−1) and epinephrine (0.60 µg kg−1). As compared with saline injection, these concentrations of collagen and epinephrine induced no significant decrease in platelet count in WT mice (P = 0.7857, n= 6) but a significant decrease in TG mice (***P < 0.0001, n= 7). Results are expressed as the mean platelet count (± SEM) per µL of blood.

Discussion

Transgenic mice overexpressing the P2Y1 receptor specifically in the platelet lineage, via the PF4 promoter, were generated with the aim of investigating the role of this receptor in several aspects of platelet activation and thrombosis. The TG mouse line obtained exhibited an 85% increase in P2Y1 receptor density on the platelet surface as compared with WT mice (Fig. 1d). Numbers of bone marrow megakaryocytes and circulating platelets were found to be within the normal ranges in TG mice (Table 1), indicating that the P2Y1 receptor does not play a significant role in platelet production. This is in agreement with earlier studies of P2Y1-deficient mice, which had platelet counts similar to those of their WT counterparts [12,13].

Platelets of TG mice overexpressing the P2Y1 receptor displayed a ‘hyperaggregatory’ response to ADP (Fig. 2a,b). Platelet aggregation induced by a low concentration of collagen was also increased (Fig. 2c), in accordance with reduced collagen responses observed in P2Y1-deficient mice [12]. These studies further support a role of the P2Y1 receptor in collagen-induced platelet aggregation. Finally, P2Y1-mediated mobilization of intracellular calcium stores was increased in TG mouse platelets (Fig. 3a), consistent with the enhanced aggregation response.

The surface density of the P2Y12 receptor remained the same on TG and WT mouse platelets (Fig. 1d), indicating that a higher level of expression of the P2Y1 gene has no repercussion on expression of the P2Y12 gene. In addition, ADP-induced inhibition of adenylyl cyclase activity was similar between the two genotypes (Fig. 3b), suggesting that overexpression of the P2Y1 receptor does not interfere with the transduction pathways associated with activation of the P2Y12 receptor.

ADP induced a significant dose-dependent secretion of granule contents in TG but not WT mouse platelets (Fig. 5a,b). This secretion is not due to the enhanced platelet aggregation as ADP-induced aggregation is maximal at 10 µmol L−1, while the secretion continued to increase in response to ADP 100 µmol L−1. As ADP (100 µmol L−1)-induced secretion of transgenic mouse platelets was inhibited by aspirin (1 mmol L−1) (Fig. 5a, lower panel), it is dependent on arachidonic acid metabolism and synthesis of TXA2. This suggests that the globally weak responses of normal platelets to ADP [15,16] would be due to the limited number of P2Y1 receptors on the cell surface rather than to a transduction pathway specific for ADP. It can be speculated that a minimum number of Gq-coupled receptors is required to induce sufficient calcium mobilization necessary to initiate arachidonic acid metabolism and TXA2 synthesis. The density of the P2Y1 receptor on WT platelets is in fact relatively low (about 150 per platelet) as compared with the densities of other receptors such as the TXA2 receptor (1500 per platelet) [18] or the thrombin receptor PAR1 (protease-activated receptor 1) (1800 per platelet) [34]. Our results therefore imply that controlled expression of the P2Y1 receptor could be important for normal platelet activation and that it would be of interest to study the regulation of its expression in humans.

The hyper-reactivity of TG mouse platelets overexpressing the P2Y1 receptor was observed not only in vitro but also in vivo, as shown by their shortened bleeding time (Fig. 2). Furthermore, these mice were more sensitive to in vivo platelet aggregation triggered by collagen and epinephrine than WT mice (Fig. 5). This correlates with the finding that mice lacking the P2Y1 receptor have increased resistance to in vivo platelet aggregation [12–14]. Moreover, in contrast to P2Y1 knockout mice, which are deficient for expression of the receptor in all tissues, mice with targeted overexpression of P2Y1 in the platelet lineage allow one to focus on the specific role of this cell surface receptor during platelet activation in vivo. Hence the global phenotype of platelet hyper-reactivity displayed by our TG mice supports the concept of the platelet P2Y1 receptor as a potential pharmacological target for new anti-thrombotic drugs.

Cases of patients presenting an alteration in platelet ADP receptors have been reported and all concern the P2Y12 receptor. These individuals display decreased platelet aggregation in response to ADP and enhanced bleeding time as the result of a severe [35,36] or moderate deficiency in P2Y12 receptor density, as yet of unknown origin [2,37,38]. Whether increased platelet P2Y1 receptor density exists in humans, and could explain some cases of platelet hyperaggregatory phenotype, remains to be investigated.

Altogether our results highlight the role of the P2Y1 receptor in hemostasis and thrombosis and the importance of tight regulation of expression of this receptor for the control of physiological and pathological processes. It remains to be assessed whether variable levels of expression of the P2Y1 receptor exist in man and whether it leads to susceptibility to the development or extension of thrombotic states.

Acknowledgements

Katya Ravid is an Established Investigator with the American Heart Association. This work was supported in part by a grant from ARMESA. The authors would like to thank Hou-Xiang Xie and Robin MacDonald for assisting in the generation of transgenic mice at BUSM, Juliette N. Mulvihill for reviewing the English of the manuscript and Monique Freund, head of the animal facility (EFS-Alsace, Strasbourg, France).

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