Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

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.

Background Essential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). Methods Phosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. Results The PS exposure on platelets in ET patients is higher than that in healthy controls. The PS‐exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time‐dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin αvβ3 on ECs, and P‐selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS‐exposed platelets were decreased after incubation with ECs. Conclusion Our results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.

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