Iloprost Concentration‐Dependently Attenuates Platelet Function and Apoptosis by Elevating PKA Activity

Iloprost, a prostacyclin (PGI₂) analogue, stimulates the IP receptor (PTGIR) to interact with the Gsα β/γ complex, leading to the activation of adenylate cyclase, which enzyme produces the second messenger cAMP. Elevation in cAMP triggers intracellular signalling events and regulates a wide variety of cellular activities. Thus, we evaluated the effects of Iloprost on platelet function and apoptosis and in vivo haemostasis and thrombosis, as well as the underlying mechanisms. Firstly, we showed that Iloprost concentration-dependently inhibited agonist-induced P-selectin exposure, integrin αIIbβ3 activation, platelet aggregation, ATP release, platelet spreading, and clot retraction. Moreover, Iloprost dose-dependently inhibited FeCl₃-induced mouse mesenteric arteriole thrombosis and markedly prolonged the tail bleeding time. Iloprost also concentration-dependently inhibited mitochondrial membrane potential (ΔΨm) depolarisation and phosphatidylserine (PS) externalisation in platelets, thereby inhibiting platelet apoptosis, and Iloprost at concentrations lower than 2 nM inhibited only platelet apoptosis but not platelet function. Importantly, Iloprost at low doses markedly elevated peripheral platelet counts in GPIbα antibody-induced immune thrombocytopenia (ITP). Mechanistic studies showed that Iloprost concentration-dependently antagonised agonist-induced decline of protein kinase A (PKA) activity and elevation of cytoplasmic Ca²⁺ in platelets, thereby attenuating platelet activation and aggregation. Elevation in PKA activity inhibited dephosphorylation of proapoptotic protein BAD and reduced caspase-3 activity, thus retarding platelet apoptosis. These data demonstrate that Iloprost dose-dependently inhibits platelet function and apoptosis by elevating PKA activity. Moderate-dose Iloprost impairs haemostasis and thrombosis via suppression of platelet function, and low-dose Iloprost elevates peripheral platelet levels by inhibiting platelet apoptosis while having no effects on platelet function.