Procoagulant Activity Of Cells Research Articles

Small molecules targeting GRP78 mitigate anti-GRP78 autoantibody–mediated tissue factor procoagulant activity in cultured endothelial cells

BackgroundThe 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor (TF) procoagulant activity (PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. ObjectivesDetermine the effects of the engagement of the anti-GRP78 autoantibody to csGRP78 on ECs and the underlying mechanisms that impact TF PCA. MethodsImmunofluorescent staining was used to determine the presence of csGRP78 in tumor necrosis factor α–treated ECs. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay (Abcam) was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind GRP78 were identified using artificial intelligence. Enzyme-linked immunosorbent assays were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. ResultsIn tumor necrosis factor α–treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by endoplasmic reticulum stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin attenuated anti-GRP78 autoantibody–induced TF PCA. We identified B07∗, as a GRP78 binder that diminished anti-GRP78 autoantibody–induced TF PCA on ECs. ConclusionThese findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

Characterization of extracellular vesicles from patients with severe aortic stenosis undergoing transcatheter aortic valve implantation and assessment of their coagulation potential

Abstract Funding Acknowledgements None. Background The pathogenesis of aortic stenosis (AS) includes processes of inflammation and calcification. Patients with aortic stenosis have increased risk of stroke. The amount of circulating extracellular vesicles (EVs) is elevated in patients with severe AS and decreases following valve intervention. The function of these EVs in the clinical setting of severe AS is not fully known. Aim With flow cytometry characterize EVs in patients with severe AS before and after Transcatheter Aortic Valve Implantation (TAVI). To investigate whether these EVs have a procoagulant effects by themselves or when incubated with human aortic endothelial cells (hAoECs), human cardiac microvascular endothelial cells (hCMVEC), human cardiac myocytes (hCM) and spheroids of hCMVEC and hCM. Methods 25 patients with severe AS were included in the study and blood samples collected before and 2 months after TAVI. The CytoFLEX flow cytometer (Beckman Coulter) was used for characterization of surface antigens on purified EVs. 2D-cultures of hAoECs, hCMVEC and hCM were incubated with EVs (100 mg/mL) for 6 hours (h) and spheroids of hCM and hCMVEC with EVs for 48h. An inhouse assay determining Tissue Factor (TF) activity by means of FXa generation in cell lysates were utilized. TF activity in purified EVs alone was determined using Tissue Factor Activity Assay kit (Human, colorimetric) from abcam. Results We found an increased level of TF positive EVs, phosphatidylserine (PS) positive EVs as well as double positive PS / TF expressing EVs in AS patients before TAVI compared to healthy controls and reduction of PS+ and TF+/PS+ expressing EVs after TAVI (Fig 1). When analyzing TF activity by means of FXa generation we found no procoagulant effect in the purified EVs alone nor when incubating EVs with hAoEC or hCM. In contrast, we found an increased TF activity in hCMVEC and spheroids of hCM and hCMVEC after incubation with EVs from AS patients compared to healthy EVs. Conclusion Patients with AS have increased amount of EVs expressing typically procoagulant surface antigens. The EVs by themselves have no procoagulant activity. However, upon interaction with hCMVEC and spheroids, consisting of hCMVEC and hCM, EVs appear to induce procoagulant activity in the recipient cells thus suggesting a possible link to thromboembolic events in coronary arteries. Additional studies are warranted to elaborate the mechanism and function of these TF, PS and TF / PS positive EVs.

OxLDL enhances procoagulant activity of endothelial cells by TMEM16F-mediated phosphatidylserine exposure.

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-inducedPS 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 significantlyinhibited 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.

Role of microbiota-derived corisin in coagulation activation during SARS-CoV-2 infection

BackgroundCoagulopathy is a major cause of morbidity and mortality in COVID-19 patients. Hypercoagulability in COVID-19 results in deep vein thrombosis, thromboembolic complications, and diffuse intravascular coagulation. Microbiome dysbiosis influences the clinical course of COVID-19. However, the role of dysbiosis in COVID-19-associated coagulopathy is not fully understood. ObjectivesThe present study tested the hypothesis that the microbiota-derived proapoptotic corisin is involved in the coagulation system activation during SARS-CoV-2 infection. MethodsThis cross-sectional study included 47 consecutive patients who consulted for symptoms of COVID-19. A mouse acute lung injury model was used to recapitulate the clinical findings. A549 alveolar epithelial, THP-1, and human umbilical vein endothelial cells were used to evaluate procoagulant and anticoagulant activity of corisin. ResultsCOVID-19 patients showed significantly high circulating levels of corisin, thrombin-antithrombin complex, D-dimer, tumor necrosis factor-α, and monocyte-chemoattractant protein-1 with reduced levels of free protein S compared with healthy subjects. The levels of thrombin-antithrombin complex, D-dimer, and corisin were significantly correlated. A monoclonal anticorisin-neutralizing antibody significantly inhibited the inflammatory response and coagulation system activation in a SARS-CoV-2 spike protein-associated acute lung injury mouse model, and the levels of corisin and thrombin-antithrombin complex were significantly correlated. In an in vitro experiment, corisin increased the tissue factor activity and decreased the anticoagulant activity of thrombomodulin in epithelial, endothelial, and monocytic cells. ConclusionThe microbiota-derived corisin is significantly increased and correlated with activation of the coagulation system during SARS-CoV-2 infection, and corisin may directly increase the procoagulant activity in epithelial, endothelial, and monocytic cells.

Butylparaben promotes phosphatidylserine exposure and procoagulant activity of human red blood cells via increase of intracellular calcium levels

Parabens are widely used as preservatives, added to products commonly used by humans, and to which individuals are exposed orally or dermally. Once absorbed into the body, parabens move into the bloodstream and travel through the systemic circulation. We investigated the potential impact of parabens on the enhanced generation of thrombin by red blood cells (RBCs), which are the principal cellular components of blood. We tested the effects of methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and p-hydroxybenzoic acid on freshly isolated human RBCs. BuP and simultaneous exposure to BuP and PrP significantly increased phosphatidylserine (PS) externalization to the outer membranes of RBCs. PS externalization by BuP was found to be mediated by increasing intracellular Ca2+ levels in RBCs. The morphological changes in BuP-treated RBCs were observed under an electron microscope. The BuP-exposed RBCs showed increased thrombin generation and adhesion to endothelial cells. Additionally, the externalization of PS exposure and thrombin generation in BuP-treated RBCs were more susceptible to high shear stress, which mimics blood turbulence under pathological conditions. Collectively, we observed that BuP induced morphological and functional changes in RBCs, especially under high shear stress, suggesting that BuP may contribute to the thrombotic risk via procoagulant activity in RBCs.

TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Temozolomide and Lomustine Induce Tissue Factor Expression and Procoagulant Activity in Glioblastoma Cells In Vitro

Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.

Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

Abstract 12521: TMEM16 Antagonists Inhibit Phosphatidylserine-Induced Procoagulant Activity in Endothelial Cells and Protect From Thrombosis in vivo

Introduction: Constitutively an anticoagulant surface, endothelial cells (ECs) switch to support coagulation following pathogenic stimuli, contributing to cardiovascular disease. To promote thrombosis, coagulation proteins must assemble on a membrane surface containing the anionic phospholipid phosphatidylserine (PS). PS is asymmetrically distributed on the inner leaflet of the plasma membrane but is rapidly externalized to the outer leaflet by calcium-activated phospholipid “scramblases”, such as TMEM16F. How phospholipid externalization on ECs supports coagulation and thrombosis has not been investigated. Hypothesis: We proposed that pharmacologic inhibition of TMEM16 phospholipid scramblases inhibits EC procoagulant activity and protects against thrombosis. Methods and Results: siRNA silencing of TMEM16F, and its closest paralog, TMEM16E, inhibited factor Xa generation and thrombin generation on the EC surface (60% and 90%, respectively, p < 0.0001), equivalent to the PS-binding protein lactadherin (100 nM). Two unrelated compounds with TMEM16 inhibitory activity, benzbromarone and CaCCinh-A01 (30 μM), prevented calcium-induced PS externalization on ECs, as determined by flow cytometry and immunofluorescence microscopy. These drugs did not inhibit intracellular calcium flux, suggesting they block the PS externalization activity of TMEM16. Benzbromarone and CaCCinh-A01 inhibited factor Xa generation on the EC surface in a dose-dependent manner (IC 50 3.2 μM and 2.0 μM, respectively). Thrombosis was evaluated using a laser injury model in the mouse cremasteric arteriole coupled with intravital microscopy to measure accumulation of fluorescently-labeled platelets and fibrin. Benzbromarone (5 mg/kg) inhibited platelet accumulation ( p < 0.05) and fibrin formation ( p < 0.01). Benzbromarone did not increase bleeding time in a tail-clip hemostasis assay. Conclusions: Activated ECs contribute anionic phospholipid to support coagulation, a process regulated by TMEM16 phospholipid scramblases. TMEM16 inhibition with benzbromarone protects from experimental thrombosis without resulting in excess bleeding. Benzbromarone is used worldwide to treat gout and could be repurposed for anti-thrombotic properties.

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes.

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.

Pb-Induced Eryptosis May Provoke Thrombosis Prior to Hemolysis

Lead (Pb) is a common metal, which can be toxic to the human body via the pollution of water or food, and can cause anemia and other diseases. However, what happens before hemolysis and anemia caused by Pb poisoning is unclear. Here, we demonstrated Pb can cause procoagulant activity of erythroid cells leading to thrombosis before hemolysis. In freshly isolated human erythroid cells, we observed that Pb resulted in hemolysis in both concentration- and time-dependent manners, but that no lysis occurred in Pb-exposed erythroid cells (≤20 μM for 1 h). Pb treatment did not cause shape changes at up to 0.5 h incubation but at 1 h incubation echinocyte and echino-spherocyte shape changes were observed, indicating that Pb can exaggerate a concentration- and time-dependent trend of shape changes in erythroid cells. After Pb treatment, ROS-independent eryptosis was shown with no increase of reactive oxygen species (ROS), but with an increase of [Ca2+]i and caspase 3 activity. With a thrombosis mouse model, we observed increased thrombus by Pb treatment (0 or 25 mg/kg). In brief, prior to hemolysis, we demonstrated Pb can cause ROS-independent but [Ca2+]i-dependent eryptosis, which might provoke thrombosis.

Hemostatic system activation in breast cancer: Searching for new biomarkers for cancer risk prediction and outcomes

In malignant diseases, the development of a systemic hypercoagulable state is the consequence of the procoagulant activity of cancer cells on the hemostatic system and based on this close relationship, alterations in the levels of hemostatic biomarkers and/or biomarkers of blood clotting activation are under investigation as a potential tool in predicting for different cancer outcomes. Today, breast cancer remains the most common tumor and the second cause of mortality for cancer in women. There is still a need for novel biomarkers for making a diagnosis at a very early stage of disease and for the classification of individuals at different risks of disease recurrence or progression. In this review, we will discuss the pathogenesis of the thrombophilic state in breast cancer patients and its interconnection with the mechanisms of malignant progression, and report on the latest results from the HYPERCAN study in the context of hemostatic biomarker development in the breast cancer setting.

Advanced cell therapy with low tissue factor loaded product NestaCell® does not confer thrombogenic risk for critically ill COVID-19 heparin-treated patients

Since the COVID-19 pandemic started, mesenchymal stromal cells (MSC) appeared as a therapeutic option to reduce the over-activated inflammatory response and promote recovery of lung damage. Most clinical studies use intravenous injection for MSC delivery, raising several concerns of thrombogenic risk due to MSC procoagulant activity (PCA) linked to the expression of tissue factor (TF/CD142). This is the first study that demonstrated procoagulant activity of TF+ human immature dental pulp stromal cells (hIDPSC, NestaCell® product) with the percentage of TF+ cells varied from 0.2% to 63.9% in plasma of healthy donors and COVID-19 heparin-treated patients. Thrombogenic risk of TF+ hIDPSCs was evaluated by rotational thromboelastometry (in vitro) and in critically ill COVID-19 patients (clinical trial). We showed that the thromboelastography is not enough to predict the risk of TF+ MSC therapies. Using TF-negative HUVEC cells, we demonstrated that TF is not a unique factor responsible for the cell's procoagulant activity. However, heparin treatment minimizes MSC procoagulant (in vitro). We also showed that the intravenous infusion of hIDPSCs with prophylactic enoxaparin administration in moderate to critically ill COVID-19 patients did not change the values of D-dimer, neither in the PT and PTT times. Our COVID-19 clinical study measured and selected the therapeutic cells with low TF (less than 25% of TF+ hIDPSCs). Our data indicate that the concomitant administration of enoxaparin and low TF-loaded is safe even for critically ill COVID-19 patients.

The α2-adrenergic receptor pathway modulating depression influences the risk of arterial thrombosis associated with BDNFVal66Met polymorphism

Depression is associated with thrombotic risk and arterial events, its proper management is strongly recommended in coronary artery disease (CAD) patients. We have previously shown that the Brain-Derived Neurotrophic Factor (BDNF)Val66Met polymorphism, related to depression, is associated with arterial thrombosis in mice, and with an increased risk of acute myocardial infarction in humans. Herein, expanding the previous findings on BDNFVal66Met polymorphism, we show that desipramine, a norepinephrine reuptake-inhibitor, rescues behavioral impairments, reduces the arterial thrombosis risk, abolishes pathological coagulation and platelet hyper-reactivity, normalizes leukocyte, platelet, and bone marrow megakaryocyte number and restores physiological norepinephrine levels in homozygous knock-in BDNF Val66Met (BDNFMet/Met) mice. The in vitro data confirm the enhanced procoagulant activity and the alpha2A-adrenergic receptor (α2A-ADR) overexpression found in BDNFMet/Met mice and we provide evidence that, in presence of Met variant, norepinephrine is crucial to up-regulate procoagulant activity and to enhance platelet generation. The α2-ADR antagonist rauwolscine rescues the prothrombotic phenotype in BDNFMet/Met mice and reduces procoagulant activity and platelet generation in cells transfected with BDNFMet plasmid or exposed to pro-BDNFMet peptide. Finally, we show that homozygous BDNFMet/Met CAD patients have hyper-reactive platelets overexpressing abundant α2A-ADR. The great proplatelet release from their megakaryocytes well reflects their higher circulating platelet number compared to BDNFVal/Val patients. These data reveal an unprecedented described role of Met allele in the dysregulation of norepinephrine/α2A-ADR pathway that may explain the predisposition to arterial thrombosis. Overall, the development of α2A-ADR inhibitors might represent a pharmacological treatment for depression-associated thrombotic conditions in this specific subgroup of CAD patients.

Hyperuricemia enhances procoagulant activity of vascular endothelial cells through TMEM16F regulated phosphatidylserine exposure and microparticle release

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.

Titanium dioxide nanoparticles enhance thrombosis through triggering the phosphatidylserine exposure and procoagulant activation of red blood cells

BackgroundExpanding biomedical application of anatase titanium dioxide (TiO2) nanoparticles (NPs) is raising the public concern on its potential health hazards. Here, we demonstrated that TiO2 NPs can increase phosphatidylserine (PS) exposure and procoagulant activity of red blood cells (RBCs), which may contribute to thrombosis.ResultsWe conducted in vitro studies using RBCs freshly isolated from healthy male volunteers. TiO2 NPs exposure (≦ 25 μg/mL) induced PS exposure and microvesicles (MV) generation accompanied by morphological changes of RBCs. While ROS generation was not observed following the exposure to TiO2 NPs, intracellular calcium increased and caspase-3 was activated, which up-regulated scramblase activity, leading to PS exposure. RBCs exposed to TiO2 NPs could increase procoagulant activity as measured by accelerated thrombin generation, and enhancement of RBC-endothelial cells adhesion and RBC-RBC aggregation. Confirming the procoagulant activation of RBC in vitro, exposure to TiO2 NPs (2 mg/kg intravenously injection) in rats increased thrombus formation in the venous thrombosis model.ConclusionCollectively, these results suggest that anatase TiO2 NPs may harbor prothrombotic risks by promoting the procoagulant activity of RBCs, which needs attention for its biomedical application.

Microvesicles, blood cells, and endothelial cells mediate phosphatidylserine-related prothrombotic state in patients with periodontitis.

Phosphatidylserine (PS) is essential for inflammation-associated thrombogenesis, but the exact effect of PS on the prothrombotic state in periodontitis is uncertain. This study aimed to determine the PS-related procoagulant state in patients with periodontitis. A total of 138 patients with periodontitis were examined compared with 42 healthy controls. PS-exposing cells and microvesicles in blood samples were detected by confocal microscopy and flow cytometry. The clotting time assay and prothrombinase complex formation assay were used to measure the procoagulant activity of microvesicles, blood cells and endothelial cells. Periodontal clinical parameters and laboratory characteristics of patients with severe periodontitis were recorded and analyzed at baseline and 6 months after non-surgical periodontal therapy. Total PS-positive (PS+ ) microvesicles and the percentage of PS+ blood cells increased in patients with severe periodontitis compared with patients with moderate/mild periodontitis or healthy controls. Endothelial cells cultured in serum from patients with severe periodontitis expressed more PS compared with those cultured in serum from healthy controls. Specifically, PS exposure on blood cells and endothelial cells significantly decreased after inhibiting the effect of inflammatory cytokines. The elevated levels of PS+ cells and microvesicles in severe periodontitis shortened clotting time and led to increased prothrombinase complex formation. Non-surgical periodontal therapy significantly attenuated the release of microvesicles and the PS exposure of blood cells in severe periodontitis. The prothrombotic state of patients with periodontitis is mediated by PS+ cells and microvesicles stimulated by elevated levels of inflammatory cytokines.

MicroRNA-340-5p inhibits endothelial apoptosis, inflammatory response, and pro-coagulation by targeting KDM4C in anti-neutrophil cytoplasmic antibody (ANCA)-mediated glomerulonephritis through activation of B cells

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a class of systemic autoimmune diseases, results in damage of various critical organs including kidneys, lungs, eyes, and nervous system. MicroRNA-340-5p was confirmed to be downregulated in autoimmune pathogenesis. However, the role of miR-340-5p remains unknown in ANCA-induced glomerulonephritis (GN). The current study aimed to explore the role of miR-340-5p in ANCA-induced GN. The animal models of ANCA-induced GN was established by experimental autoimmune vasculitis (EAV) operation. The primary glomerular endothelial cells (PGEnCs) were treated with anti-myeloperoxidase (anti-MPO) to mimic cell injury in vitro. The renal function was analysed by measuring serum creatinine, blood urea nitrogen, urine blood, urine protein and urine leukocytes. The levels of RNA and proteins were examined by RT-qPCR and western blot analysis, respectively. The binding capacity between miR-340-5p and KDM4C was detected by luciferase reporter assay. Cell apoptosis was analysed by flow cytometry in vitro. Cell viability was determined by CCK-8 assay. The cleaved caspase-3 activity was analysed by immunofluorescent assay. Cell inflammation was measured by western blot. Cell procoagulant activity was assessed by FXa generation assay. The histological changes of renal tissues were assessed by Haematoxylin and eosin (H&E) staining assay. The correlation between miR-340-5p and KDM4C level (or content of TNF-α and IL-6) was analysed by Pearson correlation analysis. The injection of anti-MPO IgG induced a significant elevation of Serum creatinine and blood urea nitrogen in serum, as well as urine blood, urine protein and urine leukocytes. Importantly, KDM4C was downregulated in model group. In mechanism, we identified that miR-340-5p bound with KDM4C 3'untranslated region (UTR), negatively regulated KDM4C in endothelial cells and negatively correlated with KDM4C in serum of GN rats. In function, we found that miR-340-5p promoted B cell activation and proliferation by downregulating KDM4C. The in vitro assays showed that the decrease of cell viability induced by anti-MPO was reversed by miR-340-5p overexpression, and further reduced by KDM4C overexpression. Inversely, the suppressive effects of miR-340-5p mimics on cell apoptosis, cleaved caspase-3 activity, inflammatory response and pro-coagulation were countervailed by KDM4C overexpression in anti-MPO-treated cells. The in vivo assays validated that miR-340-5p overexpression mitigated the impairment of renal function, and histological changes induced by anti-MPO IgG injection in model group. Finally, we found the negative correlation between miR-340-5p and TNF-α (or IL-6) content in serum of GN rats. In conclusion, we found that miR-340-5p inhibited endothelial apoptosis and inflammatory response by targeting KDM4C in ANCA-mediated GN through activation of B cells, implying a potential novel insight for treatment of ANCA-mediated GN.

Neutrophil extracellular traps induce thrombogenicity in severe carotid stenosis.

BackgroundSevere carotid stenosis is a common cause of stroke. In addition, previous clinical studies revealed that patients symptomatic of carotid stenosis suffer from increased episodes of stroke compared with their asymptomatic counterparts. However, the mechanism underlying these differences in the recurrence of stroke remains unclear.ObjectiveThe present study aimed to evaluate the levels of neutrophil extracellular traps (NETs) in the plasma of patients with severe carotid stenosis and investigate whether NETs induced procoagulant activity (PCA) in severe carotid stenosis. The study also sought to investigate the interactions between platelets or endothelial cells (ECs) and NETs.MethodsThe levels of NETs in plasma were quantified using enzyme‐linked immunosorbent assay (ELISA). In addition, NETting neutrophils and neutrophil‐platelet aggregates were detected through flow cytometry. On the other hand, the morphology of NETs formation and endothelial cells were analyzed through confocal microscopy. Finally, the procoagulant activity (PCA) of NETs and endothelial cells were assessed through ELISA and fibrin formation.ResultsPatients with symptomatic carotid stenosis patients had significantly higher levels of NETs markers compared with their asymptomatic counterparts and healthy subjects. In addition, increased levels of neutrophil‐platelet aggregates induced the generation of NETs in patients with symptomatic carotid stenosis. Moreover, NETs contributed to PCA through tissue factor (TF), in patients with carotid stenosis. Furthermore, NETs disrupted the endothelial barrier and converted endothelial cells (ECs) into PCA to enhance the PCA in patients with carotid stenosis.ConclusionsThe current study revealed differences in the levels of NETs in the plasma of symptomatic and asymptomatic patients suffering from carotid stenosis. The study also uncovered the interaction between NETs and thrombogenicity in carotid stenosis. Therefore, inhibiting NETs may be a potential biomarker and therapeutic target for recurring stroke in severe carotid stenosis.