Prostaglandin E₂ acts via the EP₄ receptor to inhibit platelet aggregation

Background

Platelets play a central role in haemostasis. Blood vessel injury leads to platelet aggregation and also invokes an inflammatory response leading to the formation of prostanoids like prostaglandin E₂ (PGE₂) and prostacyclin (PGI₂). It is known that low concentrations of PGE₂ enhance and high concentrations inhibit platelet aggregation. PGE₂ mediates its effect through four receptors: EP₁ (Gα_q signalling), EP₃ (three isoforms present; signals via G_i, G_s or G_q based on cell type), EP₂ and EP₄ (G_s signalling). PGI₂ is known to inhibit platelet aggregation through its IP receptor (G_s signalling). The role of EP₃ in exacerbating platelet aggregation has been well described. However, the role of EP₄ which acts via the same G protein coupling like IP has not been explored in detail. The aim of this study was to investigate the role of EP₄ in platelet aggregation.

Methods

Platelet aggregation assays were performed ex vivo using a platelet aggregation analyser (Aggregometer II). Blood from healthy human donors was used to obtain platelet-rich plasma. Aggregation was induced using ADP or collagen. Different agonists and antagonists were added to investigate their effects on platelet aggregation. Ca²⁺ flux changes caused by addition of agonists were also examined using a fluorescent Ca²⁺ dye (Fluo-3 AM) by flow cytometry.

Results

As expected, PGE₂ (up to 300 nM) and an EP₃ agonist (sulprostone) enhanced platelet aggregation, whereas an EP₂-selective agonist (butaprost) seemed to have no effect on platelet aggregation. On the contrary, an EP₄ agonist (ONO AE1-329) inhibited platelet aggregation in a concentration-dependent manner, and this effect could be reversed by using EP₄ antagonists (ONO AE3-208 and GW627368x) but not an IP or a DP antagonist. Inhibition of protein kinase C prevented the inhibitory effect of the EP₄ agonist, while inhibition of adenylate cyclase had no effect. The EP₄ agonist ONO AE1-329 also attenuated Ca²⁺ flux in platelets that had been stimulated with ADP.

Conclusion

These results are suggestive of an exclusive EP₄ effect on inhibition of platelet aggregation and EP₄ could be a potential target of antithrombotic therapy.