P2Y(1) and P2Y(12) receptors for ADP desensitize by distinct kinase-dependent mechanisms

Department of Microbiology & Immunology, Thomas Jefferson University, Filadelfia, Pennsylvania, United States
Blood (Impact Factor: 10.45). 06/2005; 105(9):3552-60. DOI: 10.1182/blood-2004-07-2893
Source: PubMed

ABSTRACT Adenosine 5'-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y(1) and P2Y(12). Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y(1)- and P2Y(12)-mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y(12), but not P2Y(1), desensitization is mediated by GRKs. In contrast, desensitization of P2Y(1), but not P2Y(12), is largely dependent on protein kinase C activity. This study is the first to show that both P2Y(1) and P2Y(12) desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered.

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Available from: Jiansong Luo, Sep 25, 2015
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    • "1321N1 Human astrocytoma cells stably expressing either hemagglutinin (HA)-Tagged human P2Y1 or P2Y12 receptor were Generated as previously described [5]. cells were maintained in dmem supplemented with 10% fetal bovine serum, 100 units ml−1 penicillin G, 100 µG ml−1 streptomycin sulfate and 400 µG/ML geneticin at 37°C supplemented with in a humidified atmosphere of 95% air, 5% CO2. "
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    ABSTRACT: Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.
    PLoS ONE 08/2012; 7(8):e43532. DOI:10.1371/journal.pone.0043532 · 3.23 Impact Factor
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    • "P2Y1 and P2Y12 receptors are both susceptible to receptor desensitization after prolonged agonist stimulation (Hardy et al, 2005; Mundell et al, 2006). To determine the relative importance of receptor desensitization versus ectonucleotidase activity in ADP-evoked aggregation, the pan-PKC inhibitor GF109203X was used to attenuate receptor desensitization and aggregation was measured in citrated PRP before and after recalcification (Fig 7A). "
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    ABSTRACT: ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca(2+) [(Ca(2+) )(o) ]. Lowering [Ca(2+) ](o) paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A(2) production. This study examined the role of ectonucleotidases in the [Ca(2+) ](o) -dependence of platelet activation. Reducing [Ca(2+) ](o) from millimolar to micromolar levels converted ADP (10 μmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A(2) production with aspirin had no effect on this [Ca(2+) ](o) -dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca(2+) ](o) resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca(2+) ](o) . As reported previously, thromboxane A(2) generation was enhanced at low [Ca(2+) ](o) , however this was independent of ectonucleotidase activity(.) P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y(12) receptors for sustained ADP-evoked aggregation, with a minor role for P2Y(1) . In conclusion, Ca(2+) -dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.
    British Journal of Haematology 02/2011; 153(1):83-91. DOI:10.1111/j.1365-2141.2010.08499.x · 4.71 Impact Factor
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    • "The first evidence showing the involvement of PKC in P2Y1 receptor desensitization in endothelial cells was reported in bovine pulmonary artery endothelium (Chen and Lin, 1999). In subsequent studies, PKC isoform-selective inhibitors revealed that both novel and conventional isoforms regulate P2Y1 receptor platelet activity (Hardy et al., 2005; Mundell et al., 2006). Some authors, however, suggest the influence of other protein kinases such as PKA on P2Y1 desensitization (Gainetdinov et al., 2004). "
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    ABSTRACT: Background and purpose: Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration‐dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.Experimental approach: The non‐hydrolyzable selective P2Y1 agonist ADPβS (3 µM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca2+]i were obtained in the presence and absence of inhibitors of protein kinase C (PKC).Key results: ADPβS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much‐reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium‐dependent dilatation to acetylcholine (1 µM) remained unaffected. Luminal treatment with the PKC inhibitors BIS‐I (1 µM) or BIS‐VIII (1 µM) tended to augment concentration‐dependent dilatation to ADPβS (0.1–3 µM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 µM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca2+]i in pressurized arteries confirmed the P2Y1 receptor but not M3 muscarinic receptor desensitization.Conclusions and implications: These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.
    British Journal of Pharmacology 11/2009; · 4.84 Impact Factor
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