PB0747 - Targeting Na+/K+ ATPase alpha 1 subunit is a novel anti-platelet and anti-thrombotic therapy

Background: Thrombosis is a major cause of myocardial infarction, ischemic stroke, and severe COVID-19. The sodium/potassium ATPase (NKA) is composed of α and β subunits, playing a crucial role in maintaining the sodium and potassium gradient across the cell membrane. The role of NKA on platelet function and thrombosis is unclear.

Aims: Targeting ATP1A1 (α1) is a novel anti-platelet and anti-thrombotic therapy.

Methods: Wildtype (WT) and α1 heterozygous (α1+/-) mice were used. A FeCl3-induced carotid artery injury thrombosis model, in combination with intravital microscopy, was employed for in vivo thrombosis study. Platelet transfusion assays were used to evaluate platelet α1 function on thrombosis. Western blot, co-IP, gene point mutation, etc., were used for mechanistic molecular studies.

Results: ATP1A1 heterozygosity significantly reduced its expression on platelets without affecting sodium and potassium homeostasis. Platelet α1 haplodeficiency inhibited 7.5% FeCl3-induced thrombosis in male but not in female mice (Figure 1). A similar effect was achieved by treating WT mice with ouabain (100 ng/g) or marinobufagenin (100 ng/g) for 24 hours; both bind α1 and inhibit NKA function. Transfusion of α1+/- platelets to WT mice significantly prolonged thrombosis compared to transfusion of α1+/+ platelets (Figure 2A). α1 complexed with P2Y12, which was significantly attenuated by P2Y12 LGL(121-123)SFT mutation (Figure 2B). Consequently, α1 haplodeficiency or ouabain treatment significantly inhibited ADP-induced platelet aggregation in vitro. α1 haplodeficiency or inhibition did not affect 10% FeCl3-induced thrombosis; However, it significantly prolonged thrombosis time in mice treated with a low dose of clopidogrel, which has uncertain anti-thrombotic effect when used alone (Figure 2C).

Conclusion(s): ATP1A1 binds to P2Y12 LGL motif, through which ATP1A1 plays an essential role in enhancing ADP-induced platelet activation. Targeting ATP1A1 with low-dose cardiotonic steroids ouabain and marinobufagenin significantly inhibited thrombosis. Inhibition of α1 also sensitized clopidogrel’s anti-thrombotic effect. Targeting ATP1A1 could be a novel strategy for antiplatelet and antithrombotic therapy.