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

Thrombosis is a major cause of myocardial infarction and ischemic stroke. The sodium/potassium ATPase (NKA), comprising α and β subunits, is crucial in maintaining intracellular sodium and potassium gradients. However, the role of NKA in platelet function and thrombosis remains unclear.

We utilized wild-type (WT, α1 ^+/+) and NKA α1 heterozygous (α1 ^+/-) 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 α1 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.

NKA α1 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 α1 ^+/-, but not α1 ^+/+, platelets to thrombocytopenic WT mice substantially prolonged thrombosis. Treating WT mice with low-dose ouabain or marinobufagenin, both binding NKA α1 and inhibiting its ion-transporting function, markedly inhibited thrombosis in vivo. NKA α1 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 α1 in mice or ouabain treatment of human platelets notably inhibited ADP-induced platelet aggregation. While not affecting 10% FeCl₃-induced thrombosis, NKA α1 haplodeficiency significantly prolonged thrombosis time in mice treated with an ineffective dose of clopidogrel.

NKA α1 plays an essential role in enhancing platelet activation through binding to LGL-containing platelet GPCRs. NKA α1 haplodeficiency or inhibition with low-dose ouabain and marinobufagenin significantly inhibited thrombosis and sensitized clopidogrel's anti-thrombotic effect. Targeting NKA α1 emerges as a promising antiplatelet and antithrombotic therapeutic strategy.