Supplementary Material for: Developmental Differences in Platelet Inhibition Response to Prostaglandin E1

Background: The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. Objective: To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE1). Methods: Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE1 (0–1 μM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3′5′-monophosphate (cAMP) levels (ELISA). Gαs, Gαi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. Results: Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE1 of platelet aggregation induced by ADP and collagen (PGE1 IC50: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE1-induced cAMP levels. Mechanistically, PGE1 acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the Gαs protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE1-induced cAMP levels, higher Gαs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. Conclusion: Neonatal platelets have a functionally increased PGE1-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.背景：新生儿血小板低反应性的潜在机制尚未完全阐明。既往研究已证实激动剂诱导的血小板活化存在发育性损伤，但对抑制性信号通路的差异却鲜有研究。 目的：比较前列腺素E1（prostaglandin E1, PGE1）对新生儿与成人血小板反应性的抑制作用差异。 方法：将脐带血（umbilical cord blood, CB）或成人血液制备的富血小板血浆与浓度为0~1 μM的PGE1共同孵育。采用酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测腺苷二磷酸（adenosine diphosphate, ADP）、胶原蛋白及凝血酶受体激活肽诱导的血小板聚集情况，同时检测环腺苷酸3′5′-单磷酸（cyclic adenosine 3′5′-monophosphate, cAMP）水平。分别采用免疫印迹法对成人及CB来源的超纯洗涤血小板中的Gαs、Gαi2、总蛋白激酶A（protein kinase A, PKA）及磷酸化PKA水平进行检测。 结果：与成人血小板相比，新生儿血小板对PGE1介导的ADP及胶原蛋白诱导的血小板聚集抑制作用更为敏感（ADP及胶原蛋白诱导聚集对应的PGE1半数抑制浓度（IC50）分别为14 nM与117 nM，成人组则分别为149 nM与491 nM）。新生儿血小板的基础及PGE1诱导的cAMP水平均更高。从机制上看，PGE1通过结合前列腺素类受体IP（prostacyclin receptor，前列环素受体）发挥作用，该受体与Gαs蛋白-腺苷酸环化酶通路偶联，可升高细胞内cAMP水平。cAMP可激活PKA，进而磷酸化多种下游靶抑制蛋白。与成人血小板相比，新生儿血小板的基础及PGE1诱导的cAMP水平更高，Gαs蛋白表达量也更高，且PKA依赖的蛋白磷酸化水平呈升高趋势。 结论：新生儿血小板的PGE1-cAMP-PKA通路功能活性增强。该研究结果提示，从新生儿到成人阶段抑制性信号通路的下调，可能是导致新生儿血小板低反应性的重要原因之一。