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Oxidized low‐density lipoprotein (oxLDL), a key component in atherosclerosis and hyperlipidemia, is a risk factor for atherothrombosis in dyslipidemia, yet its mechanism is poorly understood. In this study, we used oxLDL‐induced human aortic endothelial cells (HAECs) and high‐fat diet (HFD)‐fed mice as a hyperlipidemia model. Phosphatidylserine (PS) exposure, cytosolic Ca2+, reactive oxygen species (ROS), and lipid peroxidation were measured by flow cytometer. TMEM16F expression was detected by immunofluorescence, western blot, and reverse transcription polymerase chain reaction. Procoagulant activity (PCA) was measured by coagulation time, intrinsic/extrinsic factor Xase, and thrombin generation. We found that oxLDL‐induced PS exposure and the corresponding PCA of HAECs were increased significantly compared with control, which could be inhibited over 90% by lactadherin. Importantly, TMEM16F expression in oxLDL‐induced HAECs was upregulated by enhanced intracellular Ca2+ concentration, ROS, and lipid peroxidation, which led to PS exposure. Meanwhile, the knockdown of TMEM16F by short hairpin RNA significantly inhibited PS exposure in oxLDL‐induced HAECs. Moreover, we observed that HFD‐fed mice dramatically increased the progress of thrombus formation and accompanied upregulated TMEM16F expression by thromboelastography analysis, FeCl3‐induced carotid artery thrombosis model, and western blot. Collectively, these results demonstrate that TMEM16F‐mediated PS exposure may contribute to prothrombotic status under hyperlipidemic conditions, which may serve as a novel therapeutic target for the prevention of thrombosis in hyperlipidemia. Highlights: Oxidized low‐density lipoproteins (oxLDL) could increase phosphatidylserine (PS) exposure and procoagulant activity (PCA) of human aortic endothelial cells (HAECs) in a TMEM16F‐dependent manner.Increased intracellular Ca2+ and oxidative stress may contribute to TMEM16F‐mediated phospholipid scrambling.Hyperlipidemia exhibited a prothrombotic phenotype, which was likely related to TMEM16F‐mediated PS exposure on HAECs.Inhibition of TMEM16F or PS could be a novel therapeutic strategy to alleviate the thromboembolic complication of hyperlipidemia. [ABSTRACT FROM AUTHOR]

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA‐induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration‐ and time‐dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA‐treated EC, which is crucial for UA‐induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA‐treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F‐mediated PS exposure and MPs release. [ABSTRACT FROM AUTHOR]

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA. [ABSTRACT FROM AUTHOR]