UDP-sugars activate P2Y14 receptors to mediate vasoconstriction of the porcine coronary artery.

AIMS: UDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y14 receptor is a Gi/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y14 receptors are functionally expressed in the porcine coronary artery using a selective P2Y14 receptor agonist, MRS2690, and a novel selective P2Y14 receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative). METHODS AND RESULTS: Isometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y14 receptor antagonist PPTN, and the P2Y6 receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y14 receptor agonists), elicited concentration-dependent contractions of the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A2 mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y14 receptor coupling to Gi/o proteins and inhibition of adenylyl cyclase activity. CONCLUSIONS: Our data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y14 receptors.

Remote Assessment of Platelet Function in Patients with Acute Stroke or Transient Ischaemic Attack.

BACKGROUND: The TARDIS trial assessed the safety and efficacy of intensive versus guideline antiplatelet agents given for one month in patients with acute stroke or TIA. The aim of this substudy was to assess the effect of antiplatelet agents taken at baseline on platelet function reactivity and activation. METHODS: Platelet function, assessed by remotely measured surface expression of P-selectin, was assessed in patients at their time of randomisation. Data are median fluorescence values. RESULTS: The aspirin P-selectin test demonstrated that platelet expression was lower in 494 patients taking aspirin than in 162 patients not: mean 210 (SD 188) versus 570 (435), difference 360.3 (95% CI 312.2-408.4) (2p < 0.001). Aspirin did not suppress P-selectin levels below 500 units in 23 (4.7%) patients. The clopidogrel test showed that platelet reactivity was lower in 97 patients taking clopidogrel than in 585 patients not: 655 (296) versus 969 (315), difference 314.5 (95% CI 247.3-381.7) (2p < 0.001). Clopidogrel did not suppress P-selectin level below 860 units in 24 (24.7%) patients. CONCLUSIONS: Aspirin and clopidogrel suppress stimulated platelet P-selectin, although one-quarter of patients on clopidogrel have high on-treatment platelet reactivity. Platelet function testing may be performed remotely in the context of a large multicentre trial. Trial registration ISRCTN47823388.

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP.

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y12 and EP3 receptors, act as antagonists at G(i)-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G(i)-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y12 antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA2 mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI2, iloprost, PGD2, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE1 and PGE2 as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y12 antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.

Mode of action of P2Y(12 ) antagonists as inhibitors of platelet function.

P2Y(12) receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y (12)receptors. However, some P2Y(12) receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y(12) antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y(12) receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y(12) antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y(12) antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y(12) antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y (12)antagonists enhanced VASP-phosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y(12)receptors and not through another G-protein-coupled receptor.

Adenosine derived from ADP can contribute to inhibition of platelet aggregation in the presence of a P2Y12 antagonist.

OBJECTIVE: To investigate whether adenosine diphosphate (ADP)-derived adenosine might inhibit platelet aggregation, especially in the presence of a P2Y12 antagonist, where the effects of ADP at the P2Y12 receptor would be prevented. METHODS AND RESULTS: Platelet aggregation was measured in response to thrombin receptor activator peptide by platelet counting in platelet-rich plasma (PRP) and whole blood in the presence of ADP and the P2Y12 antagonists cangrelor, prasugrel active metabolite, and ticagrelor. In the presence of a P2Y12 antagonist, preincubation of PRP with ADP inhibited aggregation; this effect was abolished by adenosine deaminase. No inhibition of aggregation occurred in whole blood except when dipyridamole was added to inhibit adenosine uptake into erythrocytes. The effects of ADP in PRP and whole blood were replicated using adenosine and were directly related to changes in cAMP (assessed by vasodilator-stimulated phosphoprotein phosphorylation). All results were the same irrespective of the P2Y12 antagonist used. CONCLUSIONS: ADP inhibits platelet aggregation in the presence of a P2Y12 antagonist through conversion to adenosine. Inhibition occurs in PRP but not in whole blood except when adenosine uptake is inhibited. None of the P2Y12 antagonists studied replicated the effects of dipyridamole in the experiments that were performed.

DG-041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.