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

Abstract

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.

Prostaglandin E2Differentially Modulates Human Platelet Function through the Prostanoid EP2 and EP3 Receptors

“…The data of 17-phenyl-trinor PGE 2 (EP3 Ͼ EP1 agonist based on K i values; Kiriyama et al, 1997), mimicking 11d-16dm PGE 2 on VASP-P, microaggregates, and P-selectin expression, further support this hypothesis. Our results are consistent with and extend previous data (Heptinstall et al, 2008;Iyú et al, 2010), but we used diverse PGE 2 analogs, calculated the EC 50 values, ranked potency profiles on specific effects (enhancement of the secondary aggregation, stabilization of aggregates), and explored different platelet agonists at various concentrations. Furthermore, this is the first description of 11d-16dm PGE 2 -responsive receptors on platelets.…”

“…In our experimental setting, 11d-16dm PGE 2 not only enhanced the ADPinduced increase in intraplatelet Ca 2ϩ but also dose-dependently increased intraplatelet Ca 2ϩ when used alone. This is the first report of an EP3 agonist alone raising intraplatelet Ca 2ϩ , whereas previous studies described synergizing effects with other agonists (Heptinstall et al, 2008). It is possible that continuous recording in stopped flow is a more sensitive technique compared with flow cytometry-based single measurements.…”

“…Previous studies on mice used subthreshold, nonaggregating concentrations of ADP. More recent studies using a whole blood method based on platelet counting (Heptinstall et al, 2008) investigated a single, low ADP concentration and could not detect any direct effect of PGE 2 (0.01-10 M) on platelets, thus the role of PGE 2 was indirectly extrapolated by studying the effect of EP antagonists in PGE 2 -stimulated samples. We also showed for the first time a positive effect of PGE 2 on collagen-induced aggregation at low and intermediate collagen concentrations.…”

“…Early studies of PGE 2 showed its capacity to potentiate platelet aggregation in response to subthreshold concentrations of agonists (Shio and Ramwell, 1972;Bruno et al, 1974;Vezza et al, 1993). Upon the pharmacological characterization of EPs, EP3 receptor agonists have been shown to potentiate human platelet aggregation in response to low concentrations of various agonists (Matthews and Jones, 1993;Heptinstall et al, 2008;Iyú et al, 2010), and clinical development has begun for an EP3 antagonist (Singh et al, 2010). However, the human EP3 receptor has many splice variants that have different signaling pathways (Kotani et al, 1995;Schmid et al, 1995), and mRNAs for at least four EP3 splicing variants have been isolated from human platelets (Paul et al, 1998).…”

Novel Strategies for Assessing Platelet Reactivity

“…Platelet counting can be performed using a commercial whole blood cell counter that utilizes impedance counting technology, or using a flow cytometer [40]. The use of a fixative that stops the aggregation at the point at which a measurement is required, is an advantage if a platelet counting device is not immediately available for the counting to be performed [41,42,43].…”