Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor

Natalia Dovlatova; Yanushi D. Wijeyeratne; Susan C. Fox; Panagiotis Manolopoulos; Andrew J. Johnson; Ann E. White; M. Liaque Latif; Vera Ralevic; Stanley Heptinstall

doi:10.1160/TH07-10-0601

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2008; 100(02): 261-270
DOI: 10.1160/TH07-10-0601

Platelets and Blood Cells

Schattauer GmbH

Detection of P2Y₁₄ protein in platelets and investigation of the role of P2Y₁₄ in platelet function in comparison with the EP₃ receptor

Authors

Natalia Dovlatova^*

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
Yanushi D. Wijeyeratne^*

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
Susan C. Fox

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
Panagiotis Manolopoulos

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
Andrew J. Johnson

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
Ann E. White

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK
M. Liaque Latif

²Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, University of Nottingham, Nottingham, UK
Vera Ralevic

²Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, University of Nottingham, Nottingham, UK
Stanley Heptinstall

¹Cardiovascular Medicine, School of Medical and Surgical Sciences, University of Nottingham, Nottingham, UK

Abstract

als PDF herunterladen

Summary

mRNA encoding the recently discovered P2Y₁₄ receptor has been reported in platelets, but the presence of P2Y₁₄ receptor protein and its functionality have not been studied. If P2Y₁₄ is expressed along with P2Y₁ and P2Y₁₂ receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y₁₄ receptor in platelets and its role in platelet function. The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP₃ receptor agonist. Expression of P2Y₁₄ receptor was investigated by immunoblotting and confocal microscopy. Platelet aggregation in platelet-rich plasma (PRP) and whole blood was measured using light absorbance and platelet counting. VASP phosphorylation was investigated using flow cytometry. Immunoblotting provided evidence for P2Y₁₄ receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 µM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists. P2Y₁₄ did not substitute for P2Y₁₂ in experiments using the P2Y₁₂ antagonist AR-C69931. No effect of UDP-glucose was seen on adenylate cyclase activity as measured by VASP phosphorylation. In contrast, sulprostone acting via the EP₃ receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP₃ also partially substituted for P2Y₁₂ receptor. We have demonstrated the presence of P2Y₁₄ receptor protein in platelets, but no contribution of this receptor to several measures of platelet function has been observed. Further studies are necessary to determine whether the P2Y₁₄ receptor in platelets has any functionality.

Keywords

P2Y₁₄ - KIAA0001 - GPR105 - UDP-glucose - platelet aggregation - EP₃ - sulprostone - P2Y₁₂ - P2Y₁

PDF (1392 kb)

Referenzen

References
1 Gentry PA. The mammalian blood platelet: its role in haemostasis, inflammation and tissue repair. J Comp Pathol 1992; 107: 243-270.
2 Blann AD, Lip GY. Virchow’s triad revisited: the importance of soluble coagulation factors, the endothelium, and platelets. Thromb Res 2001; 101: 321-327.
3 Wagner DD, Burger PC. Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol 2003; 23: 2131-2137.
4 Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest 2005; 115: 3378-3384.
5 Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 1998; 50: 413-492.
6 Gachet C. Regulation of platelet functions by P2 receptors. Annu Rev Pharmacol Toxicol 2006; 46: 277-300.
7 Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev 2006; 58: 58-86.
8 Nomura N, Miyajima N, Sazuka T. et al. Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1. DNA Res 1994; 01: 47-56.
9 Abbracchio MP, Boeynaems JM, Barnard EA. et al. Characterization of the UDP-glucose receptor (renamed here the P2Y₁₄ receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci 2003; 24: 52-55.
10 Moore DJ, Murdock PR, Watson JM. et al. GPR105, a novel Gi/o-coupled UDP-glucose receptor expressed on brain glia and peripheral immune cells, is regulated by immunologic challenge: possible role in neuroimmune function. Mol Brain Res 2003; 118: 10-23.
11 Tolhurst G, Vial C, Leon C. et al. Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood 2005; 106: 1644-1651.
12 Chambers JK, Macdonald LE, Sarau HM. et al. A G protein-coupled receptor for UDP-glucose. J Biol Chem 2000; 275: 10767-10771.
13 Müller T, Bayer H, Myrtek D. et al. The P2Y₁₄ receptor of airway epithelial cells: coupling to intracellular Ca2⁺ and IL-8 secretion. Am J Respir Cell Mol Biol 2005; 33: 601-609.
14 Scrivens M, Dickenson JM. Functional expression of the P2Y₁₄ receptor in murine T-lymphocytes. Br J Pharmacol 2005; 146: 435-444.
15 Scrivens M, Dickenson JM. Functional expression of the P2Y₁₄ receptor in human neutrophils. Eur J Pharmacol 2006; 543: 166-173.
16 Lazarowski ER, Shea DA, Boucher RC. et al. Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 2003; 63: 1190-1197.
17 Skelton L, Cooper M, Murphy M. et al. Human immature monocyte-derived dendritic cells express the G protein-coupled receptor GPR105 (KIAA0001, P2Y₁₄) and increase intracellular calcium in response to its agonist, uridine diphosphoglucose. J Immunol 2003; 171: 1941-1949.
18 Matthews JS, Jones RL. Potentiation of aggregation and inhibition of adenylate cyclase in human platelets by prostaglandin E analogues. Br J Pharmacol 1993; 108: 363-369.
19 Mao GF, Jin JG, Bastepe M. et al. Prostaglandin E2 both stimulates and inhibits adenyl cyclase on platelets: comparison of effects on cloned EP4 and EP₃ prostaglandin receptor subtypes. Prostaglandins 1996; 52: 175-185.
20 Ma H, Hara A, Xiao CY. et al. Increased bleeding tendency and decreased susceptibility to thromboembolism in mice lacking the prostaglandin E receptor subtype EP(3). Circulation 2001; 104: 1176-1180.
21 Paul BZ, Ashby B, Sheth SB. Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells. Br J Haematol 1998; 102: 1204-1211.
22 Wallen NH, Ladjevardi M, Albert J. et al. Influence of different anticoagulants on platelet aggregation in whole blood; a comparison between citrate, low molecular mass heparin and hirudin. Thromb Res 1997; 87: 151-157.
23 Maayani S, Schwarz TE, Patel ND. et al. Diversity of signaling underlying responses mediated by selective activation of the platelet thromboxane A₂ (TXA₂) receptor; pharmacological and kinetic studies in vitro. Platelets 2003; 14: 359-374.
24 Maayani S, Schwarz TE, Patel ND. et al. Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y₁ receptor in vitro. Platelets 2003; 14: 445-462.
25 Heptinstall S, Anders R, Gurney M. et al. EP₃ Receptors: A new target for inhibition of platelet function in inflammatory thrombotic disease. Arterioscler Thromb Vasc Biol 2007; 27: e109.
26 Ingall AH, Dixon J, Bailey A. et al. Antagonists of the platelet P_2T receptor: a novel approach to antithrombotic therapy. J Med Chem 1999; 42: 213-220.
27 Fox SC, Sasae R, Janson S. et al. Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry. Platelets 2004; 15: 85-93.
28 Stafford NP, Pink AE, White AE. et al. Mechanisms involved in adenosine triphosphate–induced platelet aggregation in whole blood. Arterioscler Thromb Vasc Biol 2003; 23: 1928-1933.
29 Schwarz UR, Geiger J, Walter U. et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets-definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-1152.
30 Hoyjin K, Fricks I, Ivanov AA. et al. Structure-activity relationship of uridine 5’-diphosphoglucose analogues as agonists of the human P2Y₁₄ receptor. J Med Chem 2007; 50: 2030-2039.
31 Freeman K, Tsui P, Moore D. et al. Cloning, pharmacology and tissue distribution of G-protein-coupled receptor GPR105 (KIAA0001) rodent orthologs. Genomics 2001; 78: 124-128.