Selective activation of the prostaglandin E₂ receptor subtype EP₂ or EP₄ leads to inhibition of platelet aggregation

Summary

The effect of selective activation of platelet prostaglandin (PG) E₂ receptor subtype EP₂ or EP₄ on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE₂ inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE₂ on platelets lacking EP₂ (EP₂ ^–/– platelets) and EP₄ (EP₄ ^–/– platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1–259 and AE1–329, agonists for EP₂ and EP₄, respectively, potently inhibited U-46619 -induced aggregation with respective IC₅₀ values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP₂ ^–/– and EP₄ ^–/– platelets. In human platelets, AE1–259 and AE1–329 inhibited U-46619-induced aggregation with respective IC₅₀ values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1–329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1–259. AE1–329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3–208, an EP₄ antagonist. In addition, AE1–329 increased intracellular cAMP concentrations in a concentration- and EP₄-dependent manner in human platelets. These results indicate that selective activation of EP₂ or EP₄ can inhibit platelet aggregation and that EP₄ agonists are particularly promising as novel anti-platelet agents.

Keywords

Platelet aggregation - prostaglandin E₂ - EP₂ - EP₄ - anti-platelet agent

^* These authors contributed equally to this work.

• References

• 1 Ellis EF, Oelz O, Roberts LJ. 2nd, et al. Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A₂ . Science (Wash DC) 1976; 193: 1135-1137.
• 2 Needleman P, Kulkarni PS, Raz A. Coronary tone modulation: formation and actions of prostaglandins, endoperoxides, and thromboxanes. Science (Wash DC) 1977; 195: 409-412.
• 3 Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999; 340: 115-126.
• 4 Kobayashi T, Tahara Y, Matsumoto M. et al. Roles of thromboxane A₂ and prostacyclin in the development of atherosclerosis in apoE-deficient mice. J Clin Invest 2004; 114: 784-794.
• 5 Schwartz SM, Heimark RL, Majesky MW. Developmental mechanisms underlying pathology of arteries. Physiol Rev 1990; 70: 1177-1209.
• 6 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (Lond) 1993; 362: 801-809.
• 7 Eichholtz T, Jalink K, Fahrenfort I. et al. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J 1993; 291: 677-680.
• 8 Grainger DJ, Wakefield L, Bethell HW. et al. Release and activation of platelet latent TGF-β in blood clots during dissolution with plasmin. Nat Med 1995; 1: 932-937.
• 9 Rodriguez C, Gonzalez-Diez M, Badimon L. et al. Sphingosine-1-phosphate: A bioactive lipid that confers high-density lipoprotein with vasculoprotection mediated by nitric oxide and prostacyclin. Thromb Haemost 2009; 101: 665-673.
• 10 Coleman RA, Kennedy I, Humphrey PPA. et al. Prostanoids and their receptors, in Comprehensive Medicinal Chemistry Vol 3: Membranes and receptors. Emmett JC. ed 1990. Pergamon Press; Oxford, UK: pp. 643-714.
• 11 Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 1975; 72: 2994-2998.
• 12 Moncada S, Gryglewski R, Bunting S. et al. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature (Lond) 1976; 263: 663-665.
• 13 Shio H, Ramwell P. Effect of prostaglandin E 2 and aspirin on the secondary aggregation of human platelets. Nat New Biol 1972; 236: 45-46.
• 14 McDonald JW, Stuart RK. Interaction of prostaglandins E1 and E2 in regulation of cyclic-AMP and aggregation in human platelets: evidence for a common pros-taglandin receptor. J Lab Clin Med 1974; 84: 111-121.
• 15 Kobzar G, Mardla V, Järving I. et al. Comparison of the inhibitory effect of E-pros-taglandins in human and rabbit platelet-rich plasma and washed platelets. Comp Biochem Physiol C 1993; 106: 489-494.
• 16 Gray SJ, Heptinstall S. The effects of PGE₂ and CL 115,347, an antihypertensive PGE₂ analogue, on human blood platelet behaviour and vascular contractility. Eur J Pharmacol 1985; 114: 129-137.
• 17 Andersen NH, Eggerman TL, Harker LA. et al. On the multiplicity of platelet pros-taglandin receptors. I. Evaluation of competitive antagonism by aggregometry. Prostaglandins 1980; 19: 711-735.
• 18 Tynan SS, Andersen NH, Wills MT. et al. On the multiplicity of platelet prostaglandin receptors. II. The use of N-0164 for distinguishing the loci of action for PGI₂, PGD₂, PGE₂ and hydantoin analogs. Prostaglandins 1984; 27: 683-696.
• 19 Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999; 79: 1193-1226.
• 20 Sugimoto Y, Negishi M, Hayashi Y. et al. Two isoforms of the EP₃ receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with G_i proteins. J Biol Chem 1993; 268: 2712-2718.
• 21 Irie A, Sugimoto Y, Namba T. et al. Third isoform of the prostaglandin-E-receptor EP₃ subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase. Eur J Biochem 1993; 217: 313-318.
• 22 Adam M, Boie Y, Rushmore TH. et al. Cloning and expression of three isoforms of the human EP₃ prostanoid receptor. FEBS Lett 1994; 338: 170-174.
• 23 Regan JW, Bailey TJ, Donello JE. et al. Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini. Br J Pharmacol 1994; 112: 377-385.
• 24 Kotani M, Tanaka I, Ogawa Y. et al. Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP₃ subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions. Mol Pharmacol 1995; 48: 869-879.
• 25 Namba T, Sugimoto Y, Negishi M. et al. Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP₃ determines G-protein specificity. Nature (Lond) 1993; 365: 166-170.
• 26 Schmid A, Thierauch KH, Schleuning WD. et al. Splice variants of the human EP₃ receptor for prostaglandin E₂ . Eur J Biochem 1995; 228: 23-30.
• 27 Breyer RM, Bagdassarian CK, Myers SA. et al. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 2001; 41: 661-690.
• 28 Kiriyama M, Ushikubi F, Kobayashi T. et al. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 1997; 122: 217-224.
• 29 Okada Y, Hara A, Ma H. et al. Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in pros-tanoid receptors. Br J Pharmacol 2000; 131: 745-755.
• 30 Suzawa T, Miyaura C, Inada M. et al. The role of prostaglandin E receptor sub-types (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. Endocrinology 2000; 141: 1554-1559.
• 31 Shibuya I, Setiadji SV, Ibrahim N. et al. Involvement of postsynaptic EP₄ and pre-synaptic EP₃ receptors in actions of prostaglandin E₂ in rat supraoptic neurones. J Neuroendocrinol 2002; 14: 64-72.
• 32 Paul BZ, Ashby B, Sheth SB. Distribution of prostaglandin IP and EP receptor sub-types and isoforms in platelets and human umbilical artery smooth muscle cells. Br J Haematol 1998; 102: 1204-1211.
• 33 Ma H, Hara A, Xiao C-Y. et al. Increased bleeding tendency and decreased susceptibility to thromboembolism in mice lacking the prostaglandin E receptor sub-type EP₃ . Circulation 2001; 104: 1176-1180.
• 34 Fabre J-E, Nguyen M, Athirakul K. et al. Activation of the murine EP3 receptor for PGE₂ inhibits cAMP production and promotes platelet aggregation. J Clin Invest 2001; 107: 603-610.
• 35 Narumiya S, FitzGerald GA. Genetic and pharmacological analysis of prostanoid receptor function. J Clin Invest 2001; 108: 25-30.
• 36 Murata T, Ushikubi F, Matsuoka T. et al. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature (Lond) 1997; 388: 678-682.
• 37 Segi E, Sugimoto Y, Yamasaki A. et al. Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice. Biochem Biophys Res Commun 1998; 246: 7-12.
• 38 Ushikubi F, Segi E, Sugimoto Y. et al. Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP₃ . Nature (Lond) 1998; 395: 281-284.
• 39 Hizaki H, Segi E, Sugimoto Y. et al. Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP₂ . Proc Natl Acad Sci USA 1999; 96: 10501-10506.
• 40 Ushikubi F, Okuma M, Kanaji K. et al. Hemorrhagic thrombocytopathy with platelet thromboxane A₂ receptor abnormality: defective signal transduction with normal binding activity. Thromb Haemost 1987; 57: 158-164.
• 41 Schober LJ, Khandoga AL, Penz SM. et al. The EP₃-agonist sulprostone, but not prostaglandin E₂ potentiates platelet aggregation in human blood. Thromb Haemost 2010; 103: 1268-1269.