Induce Tissue Factor Expression Research Articles

TF/PAR2 Signaling Axis Supports the Protumor Effect of Neutrophil Extracellular Traps (NETs) on Human Breast Cancer Cells.

Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.

TLR3-Dependent Induction of TF Is Required to Maintain Lung Hemostasis during Poly IC-Induced ARDS-like Lung Dysfunction in Mice

Background: Respiratory virus infection causes damage to the epithelial-endothelial barrier of the lung. Mice challenged with influenza A virus (IAV) exhibit bleeding in the airspace. IAV increases tissue factor (TF) expression in epithelial cells (EpC) of the lung and deletion of lung EpC TF is associated with increased bleeding. aThe ssRNA viruses IAV undergo a dsRNA genomic phase during viral replication. Poly IC is a chemical mimetic of dsRNA. dsRNA acts like PAMPs and can activate the innate immune receptor, toll-like receptor 3 (TLR3). TLR3 stimulation of endothelial cells increased TF expression. However, the role of TLR3 in virus-induced TF expression in the lung TF has not been studied. Aim: To investigate the role of TLR3 in induction of TF during poly IC induced acute respiratory distress syndrome (ARDS)-like lung dysfunction in mice Methods: The murine lung EpC line, E10, was treated with the dsRNA mimetic (5 µg/mL), poly IC, for 0-6h. TF expression was measured by western blotting and a one-stage plasma clotting assay. Wild-type (WT) mice, mice with lung EpC TF deletion (TF fl/fl;SPCCre +, TFΔEpC) and their controls (TFfl/fl) as wells as TLR3 +/+ and TLR3 -/- mice were used in the study. To induce ARDS, poly IC (100 µg in 50 µL PBS) was administered intranasally daily for 3 days to adult mice (both sexes). Lung function was recorded noninvasively using the Buxco whole-body plethysmography system and plasma and bronchoalveolar lavage fluid (BALF) were collected before and 24 hours after the last administration of poly IC. Cellular composition of the BALF and blood was analyzed by an automated cell counter. Lung TF activity was quantified by one-stage plasma clotting and TF activity of extracellular vesicles (EV-TF) was analyzed by two-stage FXa generation assay. Results: Poly IC stimulation induced NFκB-dependent TF expression in E10 cells in dose and time-dependent manner. In addition, poly IC administration induced ARDS-like lung dysfunction, local lung inflammation and coagulation activation in WT mice compared to PBS-treated WT mice. Lung TF activity was increased 2-fold after poly IC administration compared to PBS-treated mice. Mice with lung EpC TF deficiency (TFΔEpC) did not exhibit an increase in lung TF or EV-TF in BALF after poly IC administration compared to controls. Importantly, TFΔEpC mice exhibited reduced activation of coagulation, impaired pulmonary hemostasis and increased hemorrhage into the airspace after poly IC compared to their littermate controls. In addition, TFΔEpC mice showed increased signs of poly IC-induced ARDS and had reduced lung function, increased lung injury and local inflammation compared to their control mice after poly IC. TLR3 -/- mice had significantly less EV-TF activity and less thrombin-antithrombin complexes (TAT) in BALF compared to TLR3 +/+ mice after poly IC. Conclusion: We show that the dsRNA mimetic poly IC induces lung EpC TF expression in vitro and vivo in part via TLR3-dependent NFκB pathway activation. TLR3 stimulation by intranasal poly IC results in sterile inflammation, and ARDS-like phenotype in mice similarly to that observed after IAV infection. Importantly, a deficiency in lung EpC TF leads to impaired lung hemostasis that is associated with an increased poly IC-induced ARDS phenotype as seen previously in our studies using IAV infection of mice. dsRNA induces induction of lung TF via TLR3. We proposed that induction of TF expression in the lung during respiratory viral infections protects against alveolar hemorrhage but can also contribute to thrombosis.

11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

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.

Complement component C1q initiates extrinsic coagulation via the receptor for the globular head of C1q in adventitial fibroblasts and vascular smooth muscle cells.

Vascular diseases are highly associated with inflammation and thrombosis. Elucidating links between these two processes may provide a clearer understanding of these diseases, allowing for the design of more effective treatments. The activation of complement component 1 (C1) is a crucial contributor to innate immunity and is associated with significant concentrations of circulating C1q. Many pathological pathways initiate when C1q interacts with gC1qR. This interaction plays a major role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. However, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is the more physiologically relevant coagulation arm, has not been studied. We hypothesized that C1q binding to gC1qR enhances the expression of tissue factor (TF) in adventitial fibroblastsand vascular smooth muscle cells, the primary TF bearing cells in the body. Using an enzyme-linked immunosorbent assay approach, TF expression and the role of gC1qR was observed. Cells were conditioned for 1 hwith C1q or a gC1qR blocker and C1q, to assess the role of gC1qR. Additionally, cell growth characteristics were monitored to assess changes in viability and metabolic activity. Our results indicate that the expression of TF increased significantly after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blockers and C1q exhibited no change in TF expression when compared with cells conditioned with the blocking antibodies alone. Our results show no significant differences in metabolic activity or cell viability under these conditions. This indicates that gC1qR association with C1q induces TF expression and may initiate extrinsic coagulation. Overall, this data illustrates a role for C1q in the activation of extrinsic coagulation and that gC1qR activity may link inflammation and thrombosis.

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)

Introduction: Venous thromboembolism (VTE) is an important adverse event associated with immune checkpoint inhibitors (ICI) in cancer patients (12-15% annual rate). We have developed a murine model of venous thrombosis associated with ICI therapy (referred to as ICI-CAT). We found that mice bearing CT26 colon carcinoma treated with ICI developed larger thrombi following IVC occlusion compared with mice treated with IgG. ICI did not increase thrombus weight in mice without tumors. Tissue factor (TF) plays an important role in cancer-associated thrombosis (CAT) in patients treated with conventional therapies, and TF+ extracellular vesicles (EV) were associated with the development of VTE in pancreatic cancer. However, little is known about the impact of ICI on TF expression in patients treated with ICI. To address the underlying mechanism of ICI-associated thrombosis (IAT), we have examined the expression of TF in tumor and immune cells, as well as circulating EV, in our murine CT26 model. Methods: A syngeneic colon carcinoma model (CT26) in BALB/c mice was utilized to evaluate ICI-CAT following inferior vena cava ligation (stasis). Tumor tissue was analyzed for TF expression by immunoblotting using an anti-TF antibody (ab189483, Abcam). Tumor tissue was also dissociated, and TF-positive cells were assessed by flow cytometry analysis with CD45+ and CD45- gating using antibodies for CD11b, Ly6C, Ly6G (BioLegend), and TF (R&D). EV were isolated from platelet poor plasma from ICI or control IgG-treated mice by centrifugation (20,000 x g). Procoagulant activity of EV TF was measured using a chromogenic substrate to detect FXa generation following incubation of EV with FVIIa in the presence or absence of an anti-TF antibody. Results: Treatment of tumor-bearing mice with ICI markedly induced TF expression in tumors (Fig. A, Leftupper). Furthermore, levels of EV TF antigen (Fig. A, Left bottom) and EV TF activity (Fig. A, Right) were increased in mice treated with ICI. To explore the cellular origin of TF, we performed flow cytometry with dissociated tumor cells. A greater percentage of TF-positive cells in both the CD45+ (37.8%) and CD45- (74.6%) populations were detected in tumors from ICI treated mice compared to those treated with control IgG (27.9% and 49.8%, respectively), suggesting both tumor and tumor-infiltrating immune cells contribute to the larger thrombi observed in ICI-treated mice (Fig. B). Further analysis of CD45+ TF+ cells by CD11b gating (data not shown) revealed that a TF expression in a monocyte population (CD11b+ Ly6C+) was increased 3-fold by ICI (46.6%) compared to control IgG (14.2%) (Fig. C). We also found that TF expression was increased in cultured CT26 cells in response to interferon-g, but not in response to anti-PD-1 and anti-CTLA-4 antibodies, suggesting that ICIs do not directly upregulate TF and that more complex cellular interactions and inflammatory changes may be involved. Conclusions: ICI treatment of tumor-bearing mice results in increased tumor TF expression and EV TF expression. TF was also upregulated in immune cells in the tumor microenvironment after ICI treatment, including both tumor/stromal cells (CD45-), and tumor-associated monocytes and neutrophils (CD45+). These results suggest that multiple underlying mechanisms and diverse cellular expression underlie TF expression in this model, and that these processes many contribute to the larger thrombi observed in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

SARS-CoV-2 in coronary blood from thrombus aspiration in a patient with myocardial infarction

SARS-CoV-2 in coronary blood from thrombus aspiration in a patient with myocardial infarction

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

In Covid-19 Infection, Plasma Extracellular Vesicle Tissue Factor Activity Does Not Correlate with D-Dimer Levels

Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation.Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation.Methods:Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis.Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization; n=37); intensive care (ICU; n=16); mechanical ventilation (Ventilation; n=22); or fatality (Deceased; n=22).Result:Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin; pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity.Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:<0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies.Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases. [Display omitted] DisclosuresDi Paola: CSL Behring: Consultancy, Honoraria.

Convallatoxin, the primary cardiac glycoside in lily of the valley (Convallaria majalis), induces tissue factor expression in endothelial cells.

BackgroundConvallotoxin (CNT), present in lily of the valley (Convallaria majalis), is a toxin that causes food poisoning among humans and companion animals. Although various symptoms of CNT poisoning have been well described, hypercoagulability owing to CNT is only empirically known among some veterinarians, and the underlying mechanism remains to be elucidated. CNT exerts cytotoxic effects on endothelial cells.ObjectivesThis study aimed to determine whether CNT induces the expression of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade, in endothelial cells and leads to a hypercoagulable state.MethodsHuman umbilical vein endothelial cells (HUVECs) were used for in vitro experiments. HUVECs were treated with or without CNT (50 and 100 nM) for 4 h. Phosphate‐buffered saline was used as a control. Cell viability was determined using the WST‐8 assay. Quantitative real‐time polymerase chain reaction was performed to determine TF mRNA expression. TF protein expression was observed using a laser scanning confocal microscope.ResultsThe viability of HUVECs significantly reduced after CNT treatment compared with that of non‐treated cells (p < 0.05). Moreover, a significant increase in TF mRNA and protein expression was observed after 4 h of CNT treatment. CNT elicited these effects in a dose‐dependent manner.ConclusionsTF expression induced by CNT in endothelial cells can contribute to the development of a hypercoagulable state. The present study partially revealed the mechanisms underlying the CNT‐induced hypercoagulable state. The findings can contribute to the development of a novel therapy for lily of the valley poisoning.

LPA1-mediated PKD2 activation promotes LPA-induced tissue factor expression via the p38α and JNK2 MAPK pathways in smooth muscle cells

Tissue factor (TF) is the principal initiator of blood coagulation and is necessary for thrombosis. We previously reported that lysophosphatidic acid (LPA), a potent bioactive lipid, highly induces TF expression at the transcriptional level in vascular smooth muscle cells. To date, however, the specific role of the LPA receptor is unknown, and the intracellular signaling pathways that lead to LPA induction of TF have been largely undetermined. In the current study, we found that LPA markedly induced protein kinase D (PKD) activation in mouse aortic smooth muscle cells (MASMCs). Small-interfering RNA-mediated knockdown of PKD2 blocked LPA-induced TF expression and activity, indicating that PKD2 is the key intracellular mediator of LPA signaling leading to the expression and cell surface activity of TF. Furthermore, our data reveal a novel finding that PKD2 mediates LPA-induced TF expression via the p38α and JNK2 MAPK signaling pathways, which are accompanied by the PKD-independent MEK1/2-ERK-JNK pathway. To identify the LPA receptor(s) responsible for LPA-induced TF expression, we isolated MASMCs from LPA receptor-knockout mice. Our results demonstrated that SMCs isolated from LPA receptor 1 (LPA1)-deficient mice completely lost responsiveness to LPA stimulation, which mediates induction of TF expression and activation of PKD and p38/JNK MAPK, indicating that LPA1 is responsible for PKD2-mediated activation of JNK2 and p38α. Taken together, our data reveal a new signaling mechanism in which the LPA1-PKD2 axis mediates LPA-induced TF expression via the p38α and JNK2 pathways. This finding provides new insights into LPA signaling, the PKD2 pathway, and the mechanisms of coagulation/atherothrombosis.

Interleukin-1β Induces Tissue Factor Expression in A549 Cells via EGFR-Dependent and -Independent Mechanisms

Tissue factor (TF) plays an important role in the progression and angiogenesis of tumor cells. The present study investigated the mechanism of interleukin-1β (IL-1β)-induced TF expression in A549 lung cancer cells. Based on mRNA and protein analyses, including appropriate inhibitor experiments, IL-1β was shown to induce TF expression in a time-dependent manner, mediated by IL-1 receptor-dependent phosphorylation of the mitogen-activated protein kinases (MAPK) p38, p42/44 and c-jun N-terminal kinase (JNK), as well as the Src kinase and the epidermal growth factor receptor (EGFR). Thereby, inhibition of EGFR transactivation by the Src inhibitor PP1 or direct EGFR inhibition by the EGFR tyrosine kinase inhibitor (TKI) erlotinib led to a reduction of IL-1β-induced TF expression and to a suppression of p42/44 MAPK and EGFR activation, while IL-1β-induced p38 MAPK and JNK activation remained unchanged. A knockdown of EGFR by siRNA was associated with decreased IL-1β-mediated p42/44 MAPK activation, which was no longer inhibitable by erlotinib. Concentration-dependent inhibition of IL-1β-induced TF expression was also observed in the presence of gefitinib and afatinib, two other EGFR TKIs. In summary, our results suggest that IL-1β leads to increased TF formation in lung cancer cells via both Src/EGFR/p42/44 MAPK-dependent and EGFR-independent signaling pathways, with the latter mediated via p38 MAPK and JNK.

Vascular mechanisms and manifestations of COVID-19

Vascular mechanisms and manifestations of COVID-19

Golden berry 4\u03b2-hydroxywithanolide E prevents tumor necrosis factor \u03b1-induced procoagulant activity with enhanced cytotoxicity against human lung cancer cells

Inflammation in the tumor microenvironment is positively correlated with cancer progression and metastasis as well as the risk of thromboembolism in lung cancer patients. Here we show, in human non-small cell lung cancer (NSCLC) cell lines, the master inflammatory cytokine tumor necrosis factor (TNF-α) induced tissue factor expression and procoagulant activity, and these effects were potently inhibited by 4β-hydroxywithanolide E (4HW), a natural compound isolated from Physalis peruviana. Furthermore, combination of 4HW and TNF-α caused synergistic cytotoxicity against NSCLC cells by inducing caspase-dependent apoptosis. The underlying mechanism by which 4HW reverses the procoagulant effect of TNF-α but enhances its cytotoxic effect appears to be due to inhibition of NF-κB, which is a key switch for both inflammation-induced coagulation and cell survival. Our results suggest that 4HW may have a potential application for treating inflammation-derived cancer progression and cancer-associated hypercoagulable state.

Arsenite induces tissue factor synthesis through Nrf2 activation in cultured human aortic smooth muscle cells.

Tissue factor (TF) is the initiator of the coagulation cascade, constitutively expressed in subendothelial cells such as vascular smooth muscle cells and initiating rapid coagulation when the vascular vessel is damaged. TF has been shown to be involved in the development and progression of atherosclerosis. Arsenic, an environmental pollutant, is related to the progression of atherosclerosis, although the pathogenic mechanisms are not fully elucidated. In the present study, we investigated the effect of arsenite on the expression of TF in human aortic smooth muscle cells (HASMCs) and the underlying molecular mechanisms. We found that (1) arsenite stimulated TF synthesis and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in HASMCs, (2) sulforaphane, an Nrf2 activator, also stimulated TF synthesis in HASMCs, and (3) arsenite-induced upregulation of TF synthesis was prevented by Nrf2 knockdown in HASMCs. These results suggest that arsenite promotes TF synthesis by activating the Nrf2 pathway in HASMCs and that the induction of TF expression by arsenite may be related to the progression of atherosclerosis.

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/β3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

Anti-DNA antibodies cross-reactive with β2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway

Antibodies specific for cardiolipin (CL)-β2-glycoprotein I (β2GPI) are known to induce tissue factor (TF) expression by monocytes and endothelial cells which leads to a prothrombotic state in antiphospholipid syndrome (APS), but the mechanism is not fully elucidated. Previously, we reported that the mouse monoclonal anti-CL-β2GPI antibody WB-6 cross-reacts with DNA, enters monocytes via binding to cell surface DNA, and induces TF expression. The current study aimed to identify the intracellular signaling pathways involved in this process. The binding of WB-6 to CL-β2GPI or DNA, and endocytosis was not prevented by chloroquine, but pre-treatment of the cells with chloroquine significantly suppressed TF expression. TLR9 inhibitory oligodeoxynucleotide also suppressed the WB-6-induced TF expression, suggesting a pivotal role of the TLR9 pathway in TF production. Serum antibodies obtained from a patient with APS accompanying systemic lupus erythematosus (SLE) bound to both CL-β2GPI and DNA, and induced TF in normal monocytes. This effect was suppressed by chloroquine, and abolished by removal of the DNA-binding activity. These results suggest that induction of TF expression results from TLR9 activation by DNA which was internalized together with cross-reactive antibodies produced in secondary APS accompanying SLE.

Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps.

Neutrophils through the release of neutrophil extracellular traps (NETs) containing active tissue factor (TF) are key components of thrombo-inflammation. Platelets-neutrophils interplay in ST elevation myocardial infarction (STEMI) promotes NET formation via inorganic polyphosphates (polyP) released by thrombin-activated platelets. NETs, however, are also induced by biomaterials in a platelet-independent manner. Considering the possible pleiotropic effects of Ticagrelor beyond platelet inhibition and the clinical need for novel antithrombotic strategies targeting inflammation, we investigated the effects of Ticagrelor on polyP and stent-induced NETs in STEMI. Neutrophils from healthy individuals and patients receiving Ticagrelor were stimulated with polyP or drug-eluting stents (DES) to produce NETs. To induce TF expression, neutrophils were further incubated with plasma obtained from the infarct-related artery (IRA) of STEMI patients. The effects of Ticagrelor on NETs and TF loading were assessed using fluorescence microscopy, flow cytometry, myeloperoxidase(MPO)/DNA complex ELISA, and a Western blot. Ticagrelor interrupts platelet–neutrophil interaction by attenuating NETs induced by polyP. However, Ticagrelor does not affect polyP secretion from thrombin-activated platelets. Similarly, the intracellular production of TF in neutrophils triggered by IRA plasma is not hindered by Ticagrelor. Furthermore, DES induce NETs and synchronous stimulation with IRA plasma leads to the formation of thrombogenic TF-bearing NETs. Ticagrelor inhibits stent-induced NET release. These findings suggest a novel immune-modulatory effect of Ticagrelor when it attenuates the formation of thrombogenic NETs.