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

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posted on 2019-11-29, 11:07 authored by Palma-Barqueros V., Torregrosa J.M., Caparrós-Pérez E., Mota-Pérez N., Bohdan N., Llanos M.C., Begonja A.J., Sola-Visner M., Vicente V., Teruel-Montoya R., Rivera J., Ferrer-Marín F.
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.

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