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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.