Effects of pH and concentration of sodium citrate anticoagulant on platelet aggregation measured by light transmission aggregometry induced by adenosine diphosphate.

The 2013 ISTH-SSC guidelines for the standardization of light transmission aggregometry (LTA) were largely based on expert consensus, as studies directly comparing LTA methodologies were lacking. We experimentally tested the cogency of ISTH-SSC recommendations pertaining to use of anticoagulant, in particular whether: (1) buffered citrate (BC) is preferable to unbuffered citrate (C); (2) the two recommended concentrations of sodium citrate (109 and 129 mM) are equivalent in terms of platelet aggregation (PA). Blood from 16 healthy volunteers was collected into BC and C (109 and 129 mM). PA was measured by LTA in platelet-rich plasma (PRP) stimulated by adenosine diphosphate (ADP) (2 µM) immediately after PRP preparation and up to 4 hr after blood collection; pH and platelet counts in PRP were measured in parallel. pH in PRP increased with time up to about 8 for all anticoagulants, although it was lower in BC than in C at all times. In BC, PA was lower at 45 min, but equivalent at all other times. PA was higher and more stable in sodium citrate 109 mM than in 129 mM at all times. The extent of PA did not change for up to 2 hr after blood collection, and subsequently dramatically decreased. In contrast with ISTH-SSC recommendations, (1) BC does not show advantages compared to C; (2) 109 mM citrate is preferable to 129 mM, because it better supports PA; and (3) LTA studies should be completed within 2 hr of blood collection, instead of the recommended 4 hr.

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.

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.

Detection of P2Y(14) protein in platelets and investigation of the role of P2Y(14) in platelet function in comparison with the EP(3) receptor.

mRNA encoding the recently discovered P2Y(14) receptor has been reported in platelets, but the presence of P2Y(14) receptor protein and its functionality have not been studied. If P2Y(14) is expressed along with P2Y(1) and P2Y(12) receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y(14) receptor in platelets and its role in platelet function. The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP(3) receptor agonist. Expression of P2Y(14) 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 P2Y(14) receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 muM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists. P2Y(14) did not substitute for P2Y(12) in experiments using the P2Y(12) 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 EP(3) receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP(3) also partially substituted for P2Y(12) receptor. We have demonstrated the presence of P2Y(14) 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 P2Y(14) receptor in platelets has any functionality.