Department of Pharmacology, Catholic University School of Medicine, Largo Francesco Vito 1, 00168 Rome, Italy.
J Pharmacol Exp Ther. 2011 Feb;336(2):391-402. doi: 10.1124/jpet.110.174821. Epub 2010 Nov 8.
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 ± 6 nM; E(max) of 100 ± 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE(2) stabilized reversible aggregation induced by low ADP concentrations (EC(50), 37.7 ± 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 ± 10 nM (E(max), 252 ± 51%) and 5 ± 2 nM (E(max), 300 ± 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC(50), 40 ± 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE(2) alone raised intraplatelet Ca(2+) and enhanced ADP-induced Ca(2+) increase. 11d-16dm PGE(2) and 17-phenyltrinor PGE(2) (EP3 > 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.
激活的人血小板合成前列腺素 (PG) E(2)，尽管其合成速率低于血栓烷 A(2)。PGE(2) 通过不同的受体 (EP1-4) 发挥作用，但与血栓烷相比，其在人血小板功能中的作用仍未得到充分描述。我们研究了 PGE(2) 及其类似物对体外人血小板功能和血小板及巨核细胞 EP 表达的影响。用激动剂刺激预先用 PGE(2) 或其类似物孵育的血小板，并通过光学聚集度测定法进行研究。通过停流法研究血小板内钙动员；通过流式细胞术研究血小板血管扩张刺激磷蛋白 (VASP)、P-选择素和微聚集体。PGE(2) 在纳摩尔浓度下剂量依赖性地增加 ADP 诱导的血小板聚集的次级相斜率 (速度)（EC(50)，25.6 ± 6 nM；与载体处理相比，100 ± 19% 的 E(max)增加），而不影响最终最大聚集。PGE(2) 稳定了低 ADP 浓度诱导的可逆聚集（EC(50)，37.7 ± 9 nM）。EP3 激动剂 11-去氧-16,16-二甲基 PGE(2) (11d-16dm PGE(2)) 和舒前列素增强了 ADP 诱导的聚集的次级波，其 EC(50)分别为 48.6 ± 10 nM（E(max)，252 ± 51%）和 5 ± 2 nM（E(max)，300 ± 35%）。EP2 激动剂 butaprost 抑制 ADP 诱导的次级相斜率（IC(50)，40 ± 20 nM）。EP4 刺激仅有轻微的抑制作用。11d-16dm PGE(2) 单独升高血小板内 Ca(2+)并增强 ADP 诱导的 Ca(2+)增加。在纳摩尔浓度下，11d-16dm PGE(2) 和 17-苯基三诺 PGE(2)（EP3>EP1 激动剂）拮抗 PGE(1) 诱导的 VASP 磷酸化，并诱导血小板微聚集体和 P-选择素表达。EP1、EP2、EP3 和 EP4 均在人血小板和巨核细胞上表达。PGE(2) 通过不同的 EP 精细调节人血小板的反应性。这些发现应该为基于 EP 调节的新型抗血栓形成策略的合理选择提供信息。
