Targeting P2Y14R alleviates platelet-induced NET formation and venous thrombosis through PKA/AKAP13/RhoA axis

Abstract Background and Aims Venous thromboembolism (VTE) is a disease related to high mortality and complications. Neutrophil extracellular trap (NET) formation promotes thrombo-inflammatory responses, exacerbating VTE. P2Y14 receptor (P2Y14R), which is highly expressed on neutrophils mediates NET formation, but its role and mechanism in VTE are unclear. This study aims to explore the role and mechanism of P2Y14R in VTE and to investigate the feasibility of P2Y14R-targeting therapy for VTE. Methods Venous blood of VTE patients was collected to detect the expression of P2Y14R. Deep vein thrombosis and disseminated intravascular coagulation models were developed to detect thrombus and NET formation in wild-type and neutrophil P2Y14R deficiency mice. Transcriptomics, phosphorylated proteomics, and immunofluorescence were performed to investigate the mechanisms. A high-throughput Glide docking pipeline was performed to find potent P2Y14R antagonists from repurposing drug library. Results Neutrophil P2Y14R of VTE patients was significantly increased. Neutrophil-specific P2Y14R deficiency alleviated venous thrombosis and NET formation in mice. Mechanistically, neutrophil P2Y14R deletion promotes PKA-induced AKAP13 phosphorylation, thereby inhibiting RhoA activation and cytoskeleton rearrangement, resulting in reduced neutrophil-platelet aggregates and NET release. Interestingly, proglumide was identified as a potent P2Y14R antagonist with excellent P2Y14R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on thrombosis and NET formation were verified. Conclusions Neutrophil P2Y14R deficiency regulates PKA/AKAP13/RhoA pathway to inhibit neutrophil-platelet aggregate, thereby reducing NET release and venous thrombosis. This indicates that P2Y14R may be a potential therapeutic target for the intervention of VTE using P2Y14R antagonists, including proglumide.