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

Abstract

Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thromboxane A 2 . PGE 2 acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized compared with thromboxane. We studied the effect of PGE 2 and its analogs on in vitro human platelet function and platelet and megakaryocyte EP expression. Platelets preincubated with PGE 2 or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method; platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin, and microaggregates were investigated by flow cytometry. PGE 2 at nanomolar concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC 50 , 25.6 Ϯ 6 nM; E max of 100 Ϯ 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE 2 stabilized reversible aggregation induced by low ADP concentrations (EC 50 , 37.7 Ϯ 9 nM). The EP3 agonists, 11-deoxy-16,16-dimethyl PGE 2 (11d-16dm PGE 2 ) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC 50 of 48.6 Ϯ 10 nM (E max , 252 Ϯ 51%) and 5 Ϯ 2 nM (E max , 300 Ϯ 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC 50 , 40 Ϯ 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE 2 alone raised intraplatelet Ca 2ϩ and enhanced ADP-induced Ca 2ϩ increase. 11d-16dm PGE 2 and 17-phenyltrinor PGE 2 (EP3 Ͼ EP1 agonist) at nanomolar concentrations counteracted PGE 1 -induced VASP phosphorylation and induced platelet microaggregates and P-selectin expression. EP1, EP2, EP3, and EP4 were expressed on human platelets and megakaryocytes. PGE 2 through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.

The EP3 Agonist Sulprostone Enhances Platelet Adhesion But Not Thrombus Formation Under Flow Conditions

“…Our in vitro findings that EP3 receptor stimulation enhanced washed platelet aggregation and adhesion are in line with previous findings, showing an EP3-induced increase of agonist-induced aggregation in human whole blood and PRP [18,21,25] . In addition, we found that adhesion to fibrinogen and collagen under low flow conditions is enhanced based on EP3 receptor activation; however, whole blood thrombus formation on collagen under high flow was not significantly affected by the EP3 receptor agonist sulprostone.…”

PGE2reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

“…Previous research has identified that the effects of PGE 2 on platelet function are mediated mainly through interaction with the EP3 receptor, which promotes platelet function via a G i -linked mechanism, and through interaction with the EP4 receptor, which inhibits platelet function via a G s -linked mechanism [8][9][10][11][12][13][14]. In this study, we have examined the possibility that the action of PGE 2 at EP3 and EP4 receptors can explain the previous observation that this prostaglandin is able to reverse the inhibition of aggregation by agents that stimulate cAMP production via G s , but adds to inhibition provided by agents that raise cAMP through other mechanisms, including the inhibition of cAMP PDE and direct stimulation of adenylate cyclase [15].…”

“…Another naturally occurring prostaglandin, prostaglandin E 2 (PGE 2 ), is able to raise cAMP in a similar manner by interaction with the G s -linked EP4 receptor [9][10][11][12]. However, PGE 2 is unusual in that it also interacts with a second receptor, the G i -linked EP3, to inhibit adenylate cyclase [9,13,14]. Thus, PGE 2 has the ability to both inhibit and potentiate platelet function, the resultant effect depending on the concentration of the prostaglandin and the balance between its effects at both receptors.…”

Blocking the receptor EP3 to PGE2 as a way to safely prevent atherothrombosis

“…The importance of the anti-aggregatory role of EP2 and EP4 in murine or human atherothrombosis is currently unclear, making interspecies comparisons unreliable. Conversely, in both murine and human platelets, EP3 facilitates platelet aggregation [11-14, 16, 17, 19, 20, 31, 33] and sensitizes platelets by decreasing cAMP production, even when platelets are blocked by agents such as aspirin or clopidogrel [16,17,22] . Therefore, the signaling pathway activated by EP3 translates well from murine to human platelets and, as in mice, PGE2 does facilitate the human platelet response by activating its receptor EP3.…”

“…Thus, and as opposed to the action of EP3, EP2 and EP4 activation inhibit platelet aggregation [13] . However, the relative importance of this effect is unclear both in mice [13][14][15] and humans [13,14,[16][17][18][19][20] . Murine observations revealed that the impact of PGE2 on thrombosis, i.e.…”