N-linked glycosylation of the EP3α receptor is essential for the regulation of platelet homeostasis

Abstract

Platelet activation and aggregation represent pivotal events in hemostasis and thrombosis. Prostaglandin E2 (PGE 2 ) receptor subtype 3 (EP3), the most abundantly expressed PGE 2 receptor in platelets, regulates platelet activation. Yet, the underlying mechanisms remain incompletely defined. Glycosylation, one of the most common post-translational modifications, plays a crucial role in maintaining platelet function. However, the specific contribution of EP3 glycosylation in platelets has not yet been investigated. We identified N16 and N193 of the EP3α isoform as sites for N-linked glycosylation and demonstrated that EP3α glycosylation stabilizes its capacity for G i coupling and sustains G i -mediated cAMP and Ca 2+ flux, both key modulators of platelet function. In contrast, EP3α mutants impaired U46619-, ADP- and thrombin- induced platelet aggregation and thrombin-induced clot retraction. Notably, both EP3α mutants and EP3 deficiency attenuated FeCl 3 -induced arterial thrombosis. Mechanistically, EP3α glycosylation enhanced its binding to PGE 2 and supports efficient G i coupling. The study concludes that N-linked glycosylation of EP3α is critical for platelet regulation through modulation of PGE 2 /EP3α/G i -mediated cAMP and Ca 2+ signaling.