Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl−concentration and activation of SGK1 signaling pathway

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl−levels ([Cl−]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl−]ihas a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl−]iwas increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl−]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl−]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl−]iat high levels or Cftr deficiency-induced [Cl−]iincreasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12and PAR4 expression via NF-κB signaling. Constitutively active mutant S422DSGK1 markedly increased P2Y12and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl−]iand decreased CFTR expression in CAD patients. Co-transfection of S422DSGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl−]iis a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl−]i-sensitive SGK1 signaling pathway. Therefore, [Cl−]iin platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.