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

<b><i>Background:</i></b> 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. <b><i>Objective:</i></b> To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE₁). <b><i>Methods:</i></b> 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). G_αs, G_αi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. <b><i>Results:</i></b> 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 G_αs 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 G_αs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. <b><i>Conclusion:</i></b> 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.