Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

Abstract

BackgroundThrombosis is a major cause of myocardial infarction and ischemic stroke. The sodium/potassium ATPase (NKA), comprising and {beta} subunits, is crucial in maintaining intracellular sodium and potassium gradients. However, the role of NKA in platelet function and thrombosis remains unclear. MethodsWe utilized wild-type (WT, 1+/+) and NKA 1 heterozygous (1+/-) mice, aged 8 to 16 weeks, of both sexes. An intravital microscopy-based, FeCl3-induced carotid artery injury thrombosis model was employed for in vivo thrombosis assessment. Platelet transfusion assays were used to evaluate platelet NKA 1 function on thrombosis. Human platelets isolated from healthy donors and heart failure patients treated with/without digoxin were used for platelet function and signaling assay. Complementary molecular approaches were used for mechanistic studies. ResultsNKA 1 haplodeficiency significantly reduced its expression on platelets without affecting sodium homeostasis. It significantly inhibited 7.5% FeCl3-induced thrombosis in male but not female mice without disturbing hemostasis. Transfusion of 1+/-, but not 1+/+, platelets to thrombocytopenic WT mice substantially prolonged thrombosis. Treating WT mice with low-dose ouabain or marinobufagenin, both binding NKA 1 and inhibiting its ion-transporting function, markedly inhibited thrombosis in vivo. NKA 1 formed complexes with leucine-glycine-leucine (LGL)-containing platelet receptors, including P2Y12, PAR4, and thromboxane A2 receptor. This binding was significantly attenuated by LGL>SFT mutation or LGL peptide. Haplodeficiency of NKA 1 in mice or ouabain treatment of human platelets notably inhibited ADP-induced platelet aggregation. While not affecting 10% FeCl3-induced thrombosis, NKA 1 haplodeficiency significantly prolonged thrombosis time in mice treated with an ineffective dose of clopidogrel. ConclusionNKA 1 plays an essential role in enhancing platelet activation through binding to LGL-containing platelet GPCRs. NKA 1 haplodeficiency or inhibition with low-dose ouabain and marinobufagenin significantly inhibited thrombosis and sensitized clopidogrels anti-thrombotic effect. Targeting NKA 1 emerges as a promising antiplatelet and antithrombotic therapeutic strategy.