Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y₁ receptor–null mice

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y₁ receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y₁-null mice were generated to define the role of the P2Y₁ receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y₁. These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y₁-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y₁. P2Y₁-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y₁ receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

J. Clin. Invest.104:1731–1737 (1999).

ADP, known to be secreted from cells such as erythrocytes (1), plays a key role in hemostasis by directly stimulating platelet aggregation, and through its secretion from platelet dense granules it potentiates the aggregation response induced by other agents (2, 3). Stimulation of platelets by ADP leads to a rapid influx of calcium from the external medium, the mobilization of calcium from intracellular stores and inhibition of adenylyl cyclase (3). Our understanding of the molecular basis of ADP-induced platelet activation has improved with the identification of purinoceptors involved in these processes. In previous work we showed that the metabotropic P2Y₁ receptor, originally thought to be an adenosine triphosphate (ATP) receptor (4), is in fact an ADP receptor antagonized by ATP and its analogues and is present on blood platelets (5, 6). Apart from platelets, this receptor is present in many other tissues including brain, heart, skeletal muscle, smooth muscle, endothelium, and placenta (7). The physiological roles of this widespread distribution is not known to date. However, in the cardiovascular system a critical role for extracellular nucleotides in the regulation of the vascular tone is well established (7).

The P2Y₁ receptor is necessary for ADP to induce platelet aggregation through an increase in intracellular calcium because its inhibition by selective antagonists such as adenosine-2′-phosphate-5′-phosphate (A2P5P) and adenosine-3′-phosphate-5′-phosphate (A3P5P) (8) totally abolishes ADP-induced platelet aggregation, shape change, and calcium mobilization (9–11). On the other hand, antithrombotic drugs like the thienopyridine clopidogrel or the ATP analogue AR-C66096 (12) inhibit ADP-induced platelet aggregation by acting on the adenylyl cyclase pathway, but without affecting calcium mobilization or shape change (13–17). This suggests that another P2 receptor could mediate ADP-induced inhibition of adenylyl cyclase (9–11, 13–18). Additional support for a second receptor is derived from studies of patients suffering from congenital bleeding disorders with a selective impairment of ADP-induced platelet aggregation resembling thienopyridine treatment (19, 20). These patients appear to have a deficiency of the platelet ADP receptor coupled to inhibition of adenylyl cyclase; the P2Y₁ receptor has been shown to be normal in one of the patients (21). This still-unidentified P2 receptor is thought to be coupled to a G protein of α_i2 subtype (22) and hence could belong to the P2Y metabotropic receptor family. A full aggregation response to ADP requires activation of both P2 receptors (10, 11, 23), but it is not yet clear how synergy between these 2 receptors may occur. In addition, to date no molecular entity has been reported to have the properties of a P2 receptor negatively coupled to adenylyl cyclase. Thus, doubts still remain as to whether the P2Y₁ receptor may exist in different conformations, which could account for a differential coupling to the Gq or Gi alpha subunits of G proteins. Platelets also express the ionotropic P2X₁ receptor (24–28). Its role in ADP-induced platelet aggregation is unknown and seems to be discrete, although this receptor is responsible for a unique fast calcium entry into platelets (24, 29). The aims of the present study were (a) to define the role of the P2Y₁ receptor in hemostasis and thrombosis and (b) to determine whether or not the unidentified P2 receptor coupled to inhibition of adenylyl cyclase is distinct from the P2Y₁ receptor and independently expressed in platelets. We report that targeted disruption of the P2Y₁ receptor results in impaired platelet aggregation and inhibition of acute thrombosis induced by infusion of ADP or collagen and adrenaline. We also show that platelets from P2Y₁-deficient mice are still responsive to ADP through the P2 receptor coupled to inhibition of adenylyl cyclase.

Targeted disruption of the P2Y₁ receptor gene in ES cells by homologous recombination. The coding region of the mouse P2Y₁ gene was interrupted by insertion of a neomycin resistance gene cassette (Figure 1a), and homologous recombination events were screened. A targeting frequency of 4% was observed and 2 targeted ES clones were injected into blastocysts to generate chimeric mice. Mice homozygous for either the targeted allele or the wild-type allele (Figure 1b) were obtained by interbreeding the heterozygous offspring, with the expected mendelian frequency. Two strains of P2Y₁ knockout mice were generated with identical phenotypes. These mice are viable, have no apparent morphological or physiological abnormalities, and display normal development, survival, and reproduction. The platelet count as well as other hematological parameters are identical to those of wild-type mice (Table 1).

Table 1

Hematological parameters of P2Y₁^+/+ and P2Y₁^–/– mice

Platelet aggregation is impaired in P2Y₁-deficient mice. Functional studies of platelets from P2Y₁^–/– mice demonstrated a loss of ADP-induced aggregation and shape change at concentrations of ADP up to 10 μM, confirming the necessity of this receptor for ADP-induced platelet aggregation (Figure 2). Aggregation in response to 1.25 μg/mL collagen was strongly impaired, whereas the lag time in response to 5 μg/mL collagen was markedly prolonged in P2Y₁^–/– mice (25 seconds) as compared with the wild type (15 seconds). Conversely, aggregation in response to the TXA₂ mimetic U46619 or to thrombin was affected only at subthreshold concentrations of agonists (0.5 μM and 0.01 U/mL, respectively) (Figure 2). Heterozygous mice displayed an intermediate phenotype, i.e., decreased responses to ADP and to collagen as compared with the wild-type mice, but unchanged responses to the other agonists whatever the concentration tested (data not shown).

Figure 2

In vitro aggregation responses of P2Y₁^–/– washed mice platelets as compared with wild-type. P2Y₁^–/– mice platelet aggregation is lost in response to ADP and strongly impaired in response to 1.25 μg/mL collagen, whereas the lag phase, measured from the addition of the agonist to starting of aggregation, is prolonged in response to 5 μg/mL collagen (25 seconds in P2Y₁^–/– versus 15 seconds in wild-type). In contrast, responses to thrombin and to U46619 were modified only at threshold concentrations. Bar: 1 minute.

A potentiation role of ADP in P2Y₁^–/– mice through activation of the P2 receptor negatively coupled to adenylyl cyclase. Serotonin is a weak platelet agonist known to induce shape change only through activation of the 5HT_2A receptor and mobilization of intracellular calcium, which could be used to bypass the P2Y₁ receptor (10, 23). Simultaneous addition of ADP (5 μM) and serotonin (1 μM) restored aggregation to ADP in P2Y₁^–/– platelets, suggesting the conservation of a potentiation role of ADP in P2Y₁^–/– mice. (Figure 3a). This effect was due to activation of the P2 receptor negatively coupled to adenylyl cyclase because it was completely inhibited by addition of the ATP analogue AR-C69931MX (Figure 3a). This was further confirmed by the observation that whereas the calcium response induced by ADP was abolished in the knockout mice (Figure 3b), ADP still inhibited the PGE₁-stimulated accumulation of cAMP in intact platelets (Figure 3c), an effect inhibited by AR-C69931MX. These results demonstrate the existence of an ADP receptor, coupled to the inhibition of adenylyl cyclase, which is distinct from the P2Y₁ receptor.

Figure 3

Conserved potentiating effect of ADP through activation of the P2cyc receptor. (a) Serotonin induces shape change only in both P2Y₁^+/+ and P2Y₁^–/– platelets. Simultaneous addition of serotonin and ADP restores aggregation in P2Y₁^–/– platelets, an effect inhibited by the selective P2cyc antagonist AR-C99631MX. (b) Intracellular calcium mobilization in response to ADP is lost in P2Y₁ knock-out mice. (c) ADP still inhibits cAMP formation in P2Y₁^–/– mice. Cyclic AMP levels were measured in intact washed platelets exposed to: vehicle (open bars), 1 μM PGE₁ (gray bars), PGE₁ plus 5μM ADP (black bars), PGE₁ plus ADP in the presence of 2 μM AR-C69931MX (striped bars). These data represent 1 experiment performed in duplicate, representative of 3 separate experiments giving similar results.

Platelet aggregation by activation of the P2cyc receptor requires high concentrations of ADP. Addition of 100 μM ADP to washed platelets from P2Y₁^–/– mice resulted in a slow, gradual increase of light transmission up to 20% above the baseline at 5 minutes (Figure 4a, upper-right tracing) because of formation of small platelet aggregates without optically detectable shape change. The number of platelets in the aggregates was estimated by transmission electron microscopy to be between 5 and 10 (Figure 4b, right panel). This “partial platelet aggregation” occurred without detectable calcium movements (Figure 3b) and was inhibited either by incubation with AR-C69931MX or by treatment of P2Y₁^–/– mice with clopidogrel (Figure 4a, lower-right tracing), indicating that it occurred through activation of the P2cyc receptor. Epinephrine, even at 1 mM, did not induce such a response (data not shown).

Figure 4

P2cyc receptor–mediated platelet aggregation with high concentrations of ADP. (a) Addition of 100 μM ADP to P2Y₁^–/– mice platelets induced a gradual increase of light transmission, without optically detectable shape change, up to 20% above the baseline at 5 minutes (upper-right tracing), which is inhibited by 2 μM AR-C69931MX (lower-right tracing). (b) Transmission electron microscopy of resting (left) or 100 μM ADP-induced small aggregates of 5–10 P2Y₁^–/– mice platelets (right). Bars: 0.5 μm.

P2Y₁^–/– mice have a prolonged bleeding time and a strong reduction of thrombosis. No spontaneous bleeding was observed in P2Y₁^–/– mice, but the bleeding time, which reflects in vivo primary hemostasis, was prolonged in P2Y₁^–/– mice as compared with wild-type mice. Mean bleeding time ± SD was 225.7 ± 30 seconds for P2Y₁^–/– (range 60–900 seconds, n = 43), and 126.6 ± 10 seconds for P2Y₁^+/+ (range 29–300 seconds, n = 80, with 1 mouse having a bleeding time of 800 seconds). The difference in bleeding time between the 2 populations is statistically significant (P < 0.0001, unpaired t test) (Figure 5). However, increasing doses of ADP up to 500 mg/kg infused intravenously had no effect on the platelet count in P2Y₁^–/– mice as compared with wild-type mice where platelet counts were decreased by 50% (Figure 5b), suggesting that platelets from P2Y₁^–/– mice are protected from in vivo ADP-induced platelet activation and removal from the circulation. Moreover, because collagen-induced platelet aggregation is strongly impaired, we measured the mortality induced by infusion of a mixture of collagen (0.5 mg/kg body weight) and epinephrine (60 μg/kg body weight). As shown in Figure 5c, 100% wild-type mice died within 4 minutes, whereas 50% of P2Y₁^–/– mice survived. These results demonstrate the key role of the P2Y₁ receptor in these acute thrombotic processes.

Figure 5

Hemostasis and thrombosis. (a) Bleeding time of P2Y₁^+/+ and P2Y₁^–/– mice. Each point represents 1 individual (P2Y₁^+/+, n = 43; P2Y₁^–/–, n = 80). (b) P2Y₁^–/– mice platelet counts are not modified after bolus injections of ADP whereas P2Y₁^+/+ mice platelet count is decreased by more then 50%. Each bar is the mean of 4 mice. (c) Mortality by thromboembolism in response to a bolus injection of a mixture of collagen (0.5 mg/kg body weight) and adrenaline (60 μg/kg body weight) is reduced by 50% in P2Y₁^–/– mice. Results are expressed as the percentage of the total number of animals tested.

We have generated P2Y₁ receptor–null mice in order to characterize the function of the P2Y₁ receptor in hemostasis and in thrombosis. These mice are viable, have no apparent morphological or physiological abnormalities and, so far, display normal development, survival and reproduction. This viability was somewhat surprising, since the P2Y₁ receptor is expressed early in embryonic development and throughout adult life in a wide range of tissues (7, 34). It is possible that the role of the P2Y₁ receptor is discrete in all these tissues, or that its absence is overcome by the presence of other P2 receptors (7).

In platelets, the P2Y₁ receptor has been shown to be necessary for ADP-induced aggregation (9–11), whereas a separate P2 receptor coupled to inhibition of adenylyl cyclase is thought to complete and to amplify the response (11, 14, 16, 18). However, doubts still remained as to whether the P2Y₁ receptor might be coupled to adenylyl cyclase inhibition, at least in native cells (35). Functional studies of platelets from P2Y₁^–/– mice demonstrated a loss of ADP-induced aggregation, firmly establishing the requirement for this receptor for ADP-induced platelet aggregation (Figure 2). Unexpectedly, aggregation in response to medium concentrations of collagen, able to sustain a full aggregation of wild-type platelets, was strongly impaired in P2Y₁^–/– platelets, whereas at higher concentrations the typical lag phase observed during collagen-induced aggregation was significantly prolonged (Figure 2). Collagen-induced platelet activation is a complex process involving binding of collagen to at least 2 membrane proteins, the integrin α₂β₁ and GPVI, tyrosine phosphorylation of cytosolic substrates, PLCγ2 activation, and finally generation of TXA₂ and release of ADP (36, 37). Our results suggest an unsuspected role of the P2Y₁ receptor in the early phase of collagen-induced platelet activation that will deserve further characterization. Conversely, aggregation in response to the TXA₂ mimetic U46619 or to thrombin was affected only at threshold concentrations of agonists (Figure 2). Strikingly, simultaneous addition of ADP and serotonin, used to bypass the P2Y₁ receptor (10, 23), restored aggregation in P2Y₁^–/– platelets, demonstrating the conservation of a potentiation role of ADP in P2Y₁^–/– mice (Figure 3a). This response was due to the P2cyc receptor because it was inhibited by the ATP analogue AR-C69931MX. Moreover, whereas the calcium response induced by ADP was abolished in the knock-out mice (Figure 3b), ADP still inhibited the PGE₁-stimulated accumulation of cAMP in intact platelets, this effect being blocked by AR-C69931MX (Figure 3c). These results unambiguously demonstrate the existence of the putative P2cyc (13) (or P2T_AC [14] or P2Y_AC [15, 16]) platelet ADP receptor, coupled to the inhibition of adenylyl cyclase, which is distinct from and independent of the P2Y₁ receptor.

The P2cyc receptor is believed to belong to the G protein–coupled receptor family on the basis of its coupling to the Gi₂ alpha subunit of heterotrimeric G proteins in photolabeling experiments (22) and its ability to inhibit cAMP accumulation. However, it is well-known that inhibition of adenylyl cyclase per se is not sufficient to trigger platelet aggregation (3), and agonists such as epinephrine, acting by activation of Gi only, do not promote platelet aggregation (32). However, we show here that a high concentration of ADP (100 μM) induces aggregation of P2Y₁^–/– mice platelets through activation of the P2cyc receptor (Figure 4). This effect was observed in the absence of any calcium movement as measured using fura-2/AM–loaded platelets and without the classic disc-to-sphere transformation of platelets. In contrast to ADP, epinephrine did not induce such a response even at high concentrations (not shown). Together, these results indicate that activation of the P2cyc receptor triggers additional pathways in platelets, as compared with the α_2A adrenergic receptor, leading to the formation of microaggregates. Further studies should rapidly allow the fine characterization of the intracellular pathways involved when the P2cyc receptor is activated independently of the P2Y₁ receptor.

Despite the lack of platelet aggregation in response to ADP up to 10 μM, no bleeding, either spontaneous or during delivery, was observed in P2Y₁^–/– mice, although mean bleeding time in the knockout mice was nearly twice that of the wild-type mice. P2Y₁-deficient mice displayed a strong resistance to thromboembolism induced by infusion of ADP (Figure 5b) or a mixture of collagen and epinephrine (Figure 5c) as compared with wild-type mice. These observations demonstrate that the P2Y₁ receptor must play a key role in primary hemostasis and in thrombosis. To date, antithrombotic drugs selectively inhibiting ADP-induced platelet activation act on the P2cyc receptor without affecting the P2Y₁ receptor activation. These drugs are the thienopyridine compounds ticlopidine and clopidogrel or the ATP analogues termed AR-C66096, AR-C67085, and AR-C69931MX (13–17). This study demonstrates that the P2Y₁ receptor is crucial under circumstances where collagen and ADP are the principal agents involved in platelet activation. Such circumstances are frequently encountered in small arteries where platelet interactions with the vessel wall are dependent mainly on collagen and the von Willebrand factor (38) during such clinical interventions as angioplasty, where deep injury of the vessel wall often results in vascular reocclusion (39). In addition, platelet interactions with the vessel wall are mostly dependent on extracellular nucleotides and are regulated by the presence of ectonucleotidases, among which, CD39, the ATP diphosphohydrolase, has proved to be of major importance (40, 41). Moreover, besides fibrin deposition in multiple organs because of deficiency in vascular protective effects, CD39 knock-out mice display an unexpected prolongation of the bleeding time because of selective desensitization of the P2Y₁ receptor (41). Thus, analogous to the unidentified P2 receptor sensitive to the thienopyridine clopidogrel, the P2Y₁ receptor represents a potential key pharmacological target for antithrombotic drugs. Such compounds would act in the early phase of platelet-vessel wall interactions through inhibition of ADP- and collagen-induced platelet activation. Finally, this strain of knockout mice should allow the investigation of other key roles that the P2Y₁ receptor may play, not only in the cardiovascular system, but also in all other tissues where it is expressed (7).

This work was supported by INSERM and Etablissement de Transfusion Sanguine de Strasbourg. The authors would like to thank K. Ravid, P. Chambon, S. Offermanns, and F. Lanza for helpful discussions and reading of the manuscript; D. Cassel, J. Weber, C. Schwartz, and M. Finck for expert technical assistance; and J.N. Mulvihill for reviewing the English.

Part of these data was presented at the XVIIth Congress of the International Society on Thrombosis and Haemostasis in Washington, DC, USA, August 14–21, 1999. Portions of this work have appeared in abstract form (1999 Suppl. Thromb. Haemost. p. 421).

1. Gaarder, A, Jonsen, J, Laland, S, Hellem, A, Owren, PA. Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature 1961. 192:531-532.
   View this article via: PubMed CrossRef Google Scholar
2. Born, GVR. Platelet aggregation and its reversal. Nature 1962. 194:927-929.
   View this article via: PubMed Google Scholar
3. Mills, DC. ADP receptors on platelets. Thromb Haemost 1997. 76:835-856.
   View this article via: PubMed Google Scholar
4. Webb, TE, et al. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett 1993. 324:219-225.
   View this article via: PubMed CrossRef Google Scholar
5. Léon, C, et al. The human P2Y₁ receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett 1997. 403:26-30.
   View this article via: PubMed CrossRef Google Scholar
6. Hechler, B, et al. ATP derivatives are antagonists of the P2Y₁ receptor: similarities to the platelet ADP receptor. Mol Pharmacol 1998. 53:727-733.
   View this article via: PubMed Google Scholar
7. Ralevic, V, Burnstock, G. Receptors for purines and pyrimidines. Pharmacol Rev 1998. 50:413-492.
   View this article via: PubMed Google Scholar
8. Boyer, JL, Romero-Avila, T, Schachter, JB, Harden, TK. Identification of competitive antagonists of the P2Y₁ receptor. Mol Pharmacol 1996. 50:1323-1329.
   View this article via: PubMed Google Scholar
9. Jin, J, Daniel, JL, Kunapuli, SP. Molecular basis for ADP-induced platelet activation. II. The P2Y₁ receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998. 273:2030-2034.
   View this article via: PubMed CrossRef Google Scholar
10. Savi, P, et al. Role of P2Y₁ purinoceptor in ADP-induced platelet activation. FEBS Lett 1998. 422:291-295.
    View this article via: PubMed CrossRef Google Scholar
11. Hechler, B, et al. The P2Y₁ receptor is necessary for ADP-induced platelet aggregation. Blood 1998. 92:152-159.
    View this article via: PubMed Google Scholar
12. Humphries, RG, Tomlinson, W, Ingall, AG, Cage, PA, Leff, P. FPL 66096LT: a novel, highly potent and selective antagonist at human platelet P2T-receptors. Br J Pharmacol 1994. 113:1057-1063.
    View this article via: PubMed Google Scholar
13. Hechler, B, Eckly, A, Ohlmann, P, Cazenave, JP, Gachet, C. The P2Y₁ receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel. Br J Haematol 1998. 103:858-866.
    View this article via: PubMed CrossRef Google Scholar
14. Daniel, JL, et al. Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets. J Biol Chem 1998. 273:2024-2029.
    View this article via: PubMed CrossRef Google Scholar
15. Fagura, MS, et al. P2Y₁-receptors in human platelets which are pharmacologically distinct from P2Y_ADP-receptors. Br J Pharmacol 1998. 124:157-164.
    View this article via: PubMed CrossRef Google Scholar
16. Geiger, J, Hönig-Liedl, P, Schanzenbächer, P, Walter, U. Ligand specificity and ticlopidine effects distinguish three human platelet ADP receptors. Eur J Pharmacol 1998. 351:235-246.
    View this article via: PubMed CrossRef Google Scholar
17. Geiger, J, et al. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel. Arterioscler Thromb Vasc Biol 1999. 19:2007-2011.
    View this article via: PubMed Google Scholar
18. Jantzen, H-M, et al. Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb Haemost 1999. 81:111-117.
    View this article via: PubMed Google Scholar
19. Cattaneo, M, Lecchi, A, Randi, AM, McGregor, JL, Mannuci, PM. Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood 1992. 80:2787-2796.
    View this article via: PubMed Google Scholar
20. Nurden, P, et al. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. J Clin Invest 1995. 95:1612-1622.
    View this article via: JCI PubMed Google Scholar
21. Léon, C, et al. The P2Y₁ receptor is normal in a patient presenting a severe deficiency of ADP-induced platelet aggregation. Thromb Haemost 1999. 81:775-781.
    View this article via: PubMed Google Scholar
22. Ohlmann, P, et al. The human platelet ADP receptor activates Gi₂ proteins. Biochem J 1995. 312:775-779.
    View this article via: PubMed Google Scholar
23. Jin, J, Kunapuli, SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998. 95:8070-8074.
    View this article via: PubMed CrossRef Google Scholar
24. MacKenzie, A, Mahaut-Smith, MP, Sage, SO. Activation of receptor-operated cation channels via P2X₁ not P2T purinoceptors in human platelets. J Biol Chem 1996. 271:2879-2881.
    View this article via: PubMed CrossRef Google Scholar
25. Vial, C, Hechler, B, Léon, C, Cazenave, JP, Gachet, C. Presence of P2X₁ purinoceptors in human platelets and megakaryoblastic cell lines. Thromb Haemost 1997. 78:1500-1504.
    View this article via: PubMed Google Scholar
26. Scase, TJ, Heath, MF, Allen, JM, Sage, SO, Evans, RJ. Identification of a P2X₁ purinoceptor expressed on human platelets. Biochem Biophys Res Commun 1998. 242:525-528.
    View this article via: PubMed CrossRef Google Scholar
27. Sun, B, Li, K, Okahara, K, Kambayashi, JI. P2X₁ purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression. J Biol Chem 1998. 273:11544-11547.
    View this article via: PubMed CrossRef Google Scholar
28. Clifford, EE, Parker, K, Humphreys, BD, Kertezy, SB, Dubyak, GR. The P2X₁ receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes. Blood 1998. 91:3172-3181.
    View this article via: PubMed Google Scholar
29. Takano, S, Kimura, J, Matsuoka, I, Ono, T. No requirement of P2X1 purinoceptors for platelet aggregation. Eur J Pharmacol 1999. 372:305-309.
    View this article via: PubMed CrossRef Google Scholar
30. Tokuyama, Y, Hara, M, Jones, EM, Fan, Z, Bell, GI. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun 1995. 211:211-218.
    View this article via: PubMed CrossRef Google Scholar
31. Lufkin, T, Dierich, A, LeMeur, M, Mark, M, Chambon, P. Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression. Cell 1991. 66:1105-1119.
    View this article via: PubMed CrossRef Google Scholar
32. Lanza, F, et al. Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 1998. 255:1276-1288.
33. DiMinno, G, Silver, MJ. Mouse antithrombotic assay: a simple method for the evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by ethyl alcohol. J Pharmacol Exp Ther 1983. 255:57-60.
34. Meyer, MP, Clarke, JD, Patel, K, Townsend-Nicholson, A, Burnstock, G. Selective expression of purinoceptor cP2Y₁ suggests a role for nucleotide signalling in development of the chick embryo. Dev Dyn 1999. 214:152-158.
    View this article via: PubMed CrossRef Google Scholar
35. Webb, TE, et al. The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase. Br J Pharmacol 1996. 119:1385-1392.
    View this article via: PubMed Google Scholar
36. Moroi, M, Jung, SM. Platelet receptors for collagen. Thromb Haemost 1997. 78:439-444.
    View this article via: PubMed Google Scholar
37. Watson, SP, Gibbins, J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today 1998. 19:260-264.
    View this article via: PubMed CrossRef Google Scholar
38. Savage, B, Almus-Jacobs, F, Ruggeri, ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998. 94:657-666.
    View this article via: PubMed CrossRef Google Scholar
39. Hong, MK, Mehran, R, Mintz, GS, Leon, MB. Restenosis after coronary angioplasty. Curr Probl Cardiol 1997. 22:1-36.
    View this article via: PubMed CrossRef Google Scholar
40. Robson, SC, et al. Loss of ATP diphosphohydrolase activity with endothelial cell activation. J Exp Med 1997. 185:153-163.
    View this article via: PubMed CrossRef Google Scholar
41. Enjyoji, K, et al. Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med 1999. 5:1010-1017.
    View this article via: PubMed CrossRef Google Scholar