Sodium Pump, Sex Hormones, and Thrombosis - Project Summary/Abstract Increased thrombosis risk has been observed in specific conditions, such as heart failure and sex hormone therapy, yet the underlying mechanisms remain unclear. Platelet activation and aggregation are critical components of thrombosis, though the function of many platelet proteins remain unexplored. Sodium/potassium ATPase (NKA), also known as the sodium pump, is a crucial plasma membrane protein composed of α and β subunits. NKA plays a vital role in maintaining sodium and potassium gradients, cell volume, and overall cellular homeostasis. Human cells express four α and three β subunits, forming different NKA isoforms in a tissue- or cell-specific manner. The α1 subunit is ubiquitously expressed, with proteomic studies suggesting α1β3 as the dominant isoform in platelets. However, the role of NKA in platelet function has yet to be systematically studied. Using Atp1a1 heterozygous (α1+/−) mice and a 7.5% FeCl3-induced carotid artery injury thrombosis model, we found that α1 haplodeficiency significantly inhibited thrombosis in male mice, but not in females, and reduced ADP-induced platelet aggregation in vitro. Alpha1 haplodeficiency did not alter the NKA’s pump function in platelets. Treating mice with low doses of ouabain or marinobufagenin, both potent NKA inhibitors, also markedly inhibited thrombosis in vivo. Additionally, ouabain dose-dependently inhibited ADP-induced platelet aggregation in certain individuals. Immunoprecipitation-immunoblotting assays revealed that α1 binds to P2Y12, which contains a leucine-glycine-leucine (LGL) motif that mediates the interaction of some platelet G protein-coupled receptors (GPCRs). Mutation of the LGL motif to serine- phenylalanine-threonine (SFT) significantly reduced the P2Y12-NKA α1 interaction. In WT mice, females exhibited elevated platelet α1 levels compared to males, and α1+/− mice had higher testosterone levels than their WT littermates. While gonadectomy did not affect thrombosis in female α1+/− or WT mice, it significantly enhanced thrombosis in male α1+/− mice, suggesting an interplay between NKA α1 and sex hormones in thrombosis regulation. In this project, we will test the hypothesis that α1 modulates platelet activation by fine- tuning the function of LGL-containing GPCRs, contributing to the elevated thrombosis incidence in heart failure patients and those undergoing sex hormone treatments or gonadectomy. Aim 1 will clarify how the platelet NKA α1 subunit regulates the function of LGL-containing GPCRs. Aim 2 will assess whether the NKA α1 subunit is a determining factor in the heightened thrombosis risk linked to sex hormones. This research aims to provide novel mechanistic insights into the role of NKA, cardiotonic steroids, and sex hormones in platelet biology and thrombosis, ultimately supporting the development of innovative, safer therapeutic strategies.
