ABSTRACT: Background: Hypertension is frequently accompanied by platelet hyperreactivity and an increased risk of arterial thrombosis, whereas current antiplatelet therapies provide limited benefit for primary prevention due to bleeding risks. We aimed to elucidate the specific molecular mechanism linking hypertensive conditions to platelet hyperreactivity, focusing on whether pathologically upregulated oxysterol signaling drives this residual thrombotic risk. Methods: RNA sequencing was performed on platelets isolated from hypertensive mice to identify differentially expressed genes. The expression and localization of G-protein coupled receptor 183 (GPR183) in platelets were verified using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting. Quantitative polymerase chain reaction (PCR) and Western blotting were utilized to compare platelet GPR183 expression levels between hypertensive and control mice of both sexes. Plasma 7alpha,25-dihydroxycholesterol (7alpha,25-OHC) levels in hypertensive patients and angiotensin II-infused mice were quantified via liquid chromatography-tandem mass spectrometry, and their correlation with platelet reactivity was analyzed. The influence of 7alpha,25-OHC on platelet function was systematically evaluated. Platelet activation, spreading, and clot retraction were characterized. In vivo thrombotic capacity and its pathological impact were evaluated using ferric chloride (FeCl3)-induced mesenteric artery thrombosis (monitored via intravital microscopy) and a middle cerebral artery occlusion (MCAO) model of ischemic stroke, respectively. In addition, ex vivo thrombus formation was evaluated using a microfluidic perfusion system. Whole blood was perfused over collagen-coated microchannels at physiological shear rates (1000 s-1), and thrombus area was quantified via real-time fluorescence microscopy. Mechanistic studies were further performed to elucidate the signaling pathways involved, and the therapeutic potential of pharmacological inhibition of the oxysterol-GPR183 axis on platelet activation and thrombotic events in atherosclerotic models was also investigated. Results: Initial RNA sequencing screening of hypertensive mouse platelets pointed to GPR183 as a potential candidate gene near the differential expression threshold; however, subsequent targeted validation via Quantitative polymerase chain reaction (PCR) and Western blotting robustly demonstrated its marked upregulation in hypertensive platelets at both mRNA and protein levels. Concurrently, plasma 7alpha,25-OHC levels were markedly elevated in patients with hypertension and in angiotensin II-infused hypertensive mice, and positively correlated with platelet hyperreactivity. Exposure to 7alpha,25-OHC markedly enhanced platelet activation and thrombus formation in vivo and ex vivo. Mechanistically, 7alpha,25-OHC engages platelet surface GPR183 and suppresses the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-vasodilator-stimulated phosphoprotein (VASP) signaling axis, thereby facilitating Ca2+ influx and protein kinase C (PKC) activation to potentiate platelet responsiveness. Pharmacological inhibition of this pathway, either by the selective GPR183 antagonist NIBR189 or by clotrimazole-mediated suppression of oxysterol synthesis, attenuated platelet hyperreactivity and pathological thrombosis in experimental models of hypertension without increasing bleeding risk. Conclusions: Collectively, these findings suggest that the oxysterol-GPR183 axis serves as a pathologically active amplification pathway driving platelet hyperreactivity in hypertension. Targeting this pathway may represent a potential translational strategy to selectively mitigate hypertension-related residual thrombotic risk without compromising physiological hemostasis.
