PGE2 decreases reactivity of human platelets by activating EP2 and EP4

Abstract

Introduction: Platelet hyperreactivity associates with cardiovascular events in humans. Studies in mice and humans suggest that prostaglandin E2 (PGE2) regulates platelet activation. In mice, activation of the PGE2 receptor subtype 3 (EP3) promotes thrombosis, but the significance of EP3 in humans is less well understood.

Objectives: To characterize the regulation of thromboxane-dependent human platelet activation by PGE2.

Patients/methods: Platelets collected from nineteen healthy adults were studied using an agonist of the thromboxane receptor (U46,619), PGE2, and selective agonists and/or antagonists of the EP receptor subtypes. Platelet activation was assayed by (1) optical aggregometry, (2) measurement of dense granule release, and (3) single-platelet counting.

Results: Healthy volunteers demonstrated significant interindividual variation in platelet response to PGE2. PGE2 completely inhibited U46,619-induced platelet aggregation and ATP release in 26% of subjects; the remaining 74% had partial or no response to PGE2. Antagonism of EP4 abolished the inhibitory effect of PGE2. In all volunteers, a selective EP2 agonist inhibited U46,619-induced aggregation. Furthermore, the selective EP3 antagonist DG-041 converted all PGE2 nonresponders to full responders.

Conclusions: There is significant interindividual variation of platelet response to PGE2 in humans. The balance between EP2, EP3, and EP4 activation determines its net effect. PGE2 can prevent thromboxane-induced platelet aggregation in an EP4-dependent manner. EP3 antagonism converts platelets of nonresponders to a PGE2-responsive phenotype. These data suggest that therapeutic targeting of EP pathways may have cardiovascular benefit by decreasing platelet reactivity.

Figure 1

(A) Effect of preincubation with 100 nM PGE₂ before U46,619-induced aggregation among PGE₂ responders. PGE₂ significantly inhibited aggregation in this subgroup of healthy volunteers (** P < 0.001 for submaximal U46,619, n=5; * P < 0.005 for maximal U46,619, n=3; paired t-test). (B) Representative aggregation traces from a PGE₂ responder, partial responder, and nonresponder. Aggregation was induced with submaximal concentrations of U46,619. PGE₂ (100nM) was added 30 seconds before U46,619. Upper curves: aggregation. Lower curves: ATP release. (C) Dose-response relationships for U46,619-induced aggregation and (D) ATP release in human PRP for both responders and nonresponders showing no difference in the dose-response to U46,619 between these groups. Bars represent mean ± SEM.

Figure 2

Pre-incubation with PGE₂ (100 nm) inhibited platelet dense granule release (A) and increased the number of single platelets remaining after the addition of agonist (B) among PGE₂ responders but not among PGE₂ nonresponders. (** P<0.005, n=5; *P < 0.05, n=4; paired t-test).

Figure 3

Pretreatment with the EP4 antagonist MF-191 (50nM) blocked the inhibitory effect of PGE₂ among responders (n=4). Aggregation was induced with submaximal concentrations of U46,619. Bars represent mean ± SEM. (P<0.0001 by repeated measures ANOVA; ** P<0.001, compared with all other conditions by Newman-Keuls multiple comparison test.)

Figure 4

EP3 antagonist converted nonresponders to a responsive phenotype that requires EP4 (n = 6). Human PRP from PGE₂ nonresponders was pretreated with the EP3 antagonist DG-041 (1 μM) and/or the EP4 antagonist MF-191 (50 nM) before the addition of PGE₂ (100 nM) and the subsequent induction of aggregation with submaximal concentrations of U46,619. (P<0.0001 by repeated measures ANOVA; ** P < 0.001 compared with all other conditions by Newman-Keuls multiple comparison test.)

Figure 5

(A) The EP2 agonist butaprost free acid (~250 nM) inhibited aggregation of human PRP induced by submaximal concentrations of U46,619. Co-incubation with the EP3 agonist sulprostone (~25nM) masked this effect. (P<0.0001 by repeated measures ANOVA, n = 8; ** P<0.001, compared with other conditions, * P>0.05 compared with control, by Newman-Keuls multiple comparison test.) (B) Sulprostone potentiated aggregation induced by subthreshold concentrations of U46,619 (~200 nM). Co-incubation with the EP4 agonist PGE₁-OH partially attenuated this potentiation. (P<0.0001 by repeated measures ANOVA, n = 9; ** P < 0.001 compared with other conditions, * P < 0.05 compared with sulprostone by Newman-Keuls multiple comparison test.)