Increased Tissue Factor Expression Research Articles

E-Cigarettes induce expression of procoagulant tissue factor in cultivated human endothelial cells.

E-cigarettes (ECIG) are proposed as an alternative for regular tobacco users with less dangerous effects for health. Several studies demonstrated that ECIG exert deleterious cardiovascular effects and promote platelet dependent thrombosis. However, ECIG role on Tissue Factor-dependent thrombosis is still unknown. Dysfunctional endothelial cells (ECs) are known to express Tissue Factor (TF) on their surface. Aim of the present study was to investigate whether ECIG might promote TF expression in ECs, shifting them to a pro thrombotic phenotype. Human Umbilical Vein Endothelial Cells (HUVEC) were incubated with increasing doses of ECIG (commercially available and mix of propylene glycol/vegetable glycerine/nicotine 18mg/mL) up to 1.8mg/mL. TF gene expression and protein levels were assessed at different time points by Real Time PCR and Western Blot, respectively. TF surface expression and activity were also measured by FACS analysis and coagulation assay. Finally, NF-kB translocation was investigated as possible mechanism of action. Potential protective effects by Rosuvastatin were also investigated. ECIG significantly increased TF expression at both gene and protein levels in a time and dose dependent manner. Surface expression and procoagulant activity were increased as well. These phenomena appeared modulated by the NF-κB pathway. Rosuvastatin reduced ECIG effects on TF-mRNA. Although in vitro, we indicate that ECIG promote a pro thrombotic phenotype in ECs via expression of functional TF. Data of the present study permit to shed a brighter light on the still partially unresolved issue about the role of ECIG in development of cardiovascular diseases suggesting that they might represent a potential risk factor for thrombotic cardiovascular events.

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.

Presence of procoagulant peripheral blood mononuclear cells in severe COVID-19 patients relate to ventilation perfusion mismatch and precede pulmonary embolism

PurposePulmonary emboli (PE) contribute substantially to coronavirus disease 2019 (COVID-19) related mortality and morbidity. Immune cell-mediated hyperinflammation drives the procoagulant state in COVID-19 patients, resulting in immunothrombosis. To study the role of peripheral blood mononuclear cells (PBMC) in the procoagulant state of COVID-19 patients, we performed a functional bioassay and related outcomes to the occurrence of PE. Secondary aims were to relate this functional assay to plasma D-dimer levels, ventilation perfusion mismatch and TF expression on monocyte subsets. MethodsPBMC from an ICU biobank were obtained from 20 patients with a computed tomography angiograph (CTA) proven PE and compared to 15 COVID-19 controls without a proven PE. Functional procoagulant properties of PBMC were measured using a modified fibrin generation time (MC-FGT) assay. Tissue factor (TF) expression on monocyte subsets were measured by flow cytometry. Additional clinical data were obtained from patient records including end-tidal to arterial carbon dioxide gradient. ResultsMC-FGT levels were highest in the samples taken closest to the PE detection, similar to the end-tidal to arterial carbon dioxide gradient (ETCO2 – PaCO2), a measurement to quantify ventilation-perfusion mismatch. In patients without proven PE, peak MC-FGT relates to an increase in end-tidal to arterial carbon dioxide gradient. We identified non-classical, CD16 positive monocytes as the subset with increased TF expression. ConclusionWe show that the procoagulant state of PBMC could aid in early detection of PE in COVID-19 ICU patients. Combined with end-tidal to ETCO2 – PaCO2 gradient, these tests could improve early detection of PE on the ICU.

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.

The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.

Syndecan-1 (Sdc-1) upregulation is associated with poor prognosis in breast cancer. Sdc-1 knockdown results in reduced angiogenesis and the dysregulation of tissue factor (TF) pathway constituents. Here, we evaluate the regulatory mechanisms and functional consequences of the Sdc-1/TF-axis using Sdc-1 knockdown and overexpression approaches in MCF-7 and MDA-MB-231 breast cancer cells. Gene expression was analyzed by means of qPCR. Thrombin generation and cell migration were detected. Cell-cycle progression and apoptosis were investigated using flow cytometry. In MDA-MB-231 cells, IL6, IL8, VEGF, and IGFR-dependent signaling affected TF pathway expression depending on Sdc-1. Notably, Sdc-1 depletion and TF pathway inhibitor (TFPI) synergistically affected PTEN, MAPK, and STAT3 signaling. At the functional level, the antiproliferative and pro-apoptotic effects of TFPI depended on Sdc-1, whereas Sdc-1's modulation of cell motility was not affected by TFPI. Sdc-1 overexpression in MCF-7 and MDA-MB-231 cells led to increased TF expression, inducing a procoagulative phenotype, as indicated by the activation of human platelets and increased thrombin formation. A novel understanding of the functional interplay between Sdc-1 and the TF pathway may be compatible with the classical co-receptor role of Sdc-1 in cytokine signaling. This opens up the possibility of a new functional understanding, with Sdc-1 fostering coagulation and platelet communication as the key to the hematogenous metastatic spread of breast cancer cells.

KRAS mutations in pancreatic cancer: could we talk about a risk factor for the development of thromboembolic phenomena?

The oncogenic KRAS mutation is associated with increased tissue factor expression and thus hypercoagulability. In this regard, numerous studies published in the last decade have shown that KRAS mutations are an important risk factor for the development of thromboembolic phenomena in neoplasms of the digestive tract, such as colorectal cancer. On the other hand, some recently published studies suggest that KRAS mutations are also associated with an increased risk of developing thromboembolic phenomena in pancreatic cancer. Based on these premises, we have conducted a single-centre retrospective study on a cohort of patients with pancreatic cancer. Our aim is to demonstrate whether there is an association between the presence of KRAS mutations in our cohort of pancreatic cancer patients and an increased risk of developing thromboembolic phenomena.

High Plasma Levels of Activated Factor VII-Antithrombin Complex Point to Increased Tissue Factor Expression in Patients with SARS-CoV-2 Pneumonia: A Potential Link with COVID-19 Prothrombotic Diathesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of coronavirus disease 2019 (COVID-19), in which coagulation abnormalities and endothelial dysfunction play a key pathogenic role. Tissue factor (TF) expression is triggered by endothelial dysfunction. Activated factor VII-antithrombin (FVIIa-AT) complex reflects indirectly FVIIa-TF interaction and has been proposed as a potential biomarker of prothrombotic diathesis. FVIIa-AT plasma concentration was measured in 40 patients (30 males and 10 females; 64.8 ± 12.3 years) admitted with SARS-CoV-2 pneumonia during the first pandemic wave in Italy. Two sex- and age-matched cohorts without COVID-19, with or without signs of systemic inflammation, were used to compare FVIIa-AT data. The FVIIa-AT plasma levels in COVID-19 patients were higher than those in non-COVID-19 subjects, either with or without inflammation, while no difference was observed among non-COVID-19 subjects. The association between COVID-19 and FVIIa-AT levels remained significant after adjustment for sex, age, C-reactive protein, renal function, fibrinogen, prothrombin time and activated partial thromboplastin time. Our results indicate that SARS-CoV-2 infection, at least during the first pandemic wave, was characterized by high FVIIa-AT levels, which may suggest an enhanced FVIIa-TF interaction in COVID-19, potentially consistent with SARS-CoV-2-induced endotheliopathy.

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Abstract Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P < 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P < 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P < 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P < 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P < 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P < 0.01; CLI-095: −100 ± 6,6 %, n = 6, P < 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P < 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p<0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p<0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p<0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p<0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p<0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p<0.01; CLI-095: −100±6,6%, n=6, p<0.01; BAY-11-7082: −100±2,1%, n=6, p<0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. Funding Acknowledgement Type of funding sources: None.

Thrombolome and Its Emerging Role in Chronic Kidney Diseases.

Patients with chronic kidney disease (CKD) are at an increased risk of thromboembolic complications, including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. These complications lead to increased mortality. Evidence points to the key role of CKD-associated dysbiosis and its effect via the generation of gut microbial metabolites in inducing the prothrombotic phenotype. This phenomenon is known as thrombolome, a panel of intestinal bacteria-derived uremic toxins that enhance thrombosis via increased tissue factor expression, platelet hyperactivity, microparticles release, and endothelial dysfunction. This review discusses the role of uremic toxins derived from gut-microbiota metabolism of dietary tryptophan (indoxyl sulfate (IS), indole-3-acetic acid (IAA), kynurenine (KYN)), phenylalanine/tyrosine (p-cresol sulfate (PCS), p-cresol glucuronide (PCG), phenylacetylglutamine (PAGln)) and choline/phosphatidylcholine (trimethylamine N-oxide (TMAO)) in spontaneously induced thrombosis. The increase in the generation of gut microbial uremic toxins, the activation of aryl hydrocarbon (AhRs) and platelet adrenergic (ARs) receptors, and the nuclear factor kappa B (NF-κB) signaling pathway can serve as potential targets during the prevention of thromboembolic events. They can also help create a new therapeutic approach in the CKD population.

Anaplastic lymphoma kinase rearrangement may increase the incidence of venous thromboembolism by increasing tissue factor expression in advanced lung adenocarcinoma.

BackgroundPatients with lung cancer are at an increased risk for venous thromboembolism (VTE). Approximately 8–15% of patients with advanced non-small-cell lung cancer (NSCLC) experience a VTE throughout the course of the disease. However, the incidence of VTE in different NSCLC molecular subtypes is rarely reported, although there are significant differences in clinical feature and prognosis. Tissue factor (TF) expressed in many solid tumors could trigger the downstream coagulation cascade and lead to thrombin generation and clot formation.MethodsIn the present study, retrospective data were obtained from electronic medical records at Henan Cancer Hospital in China between January 2015 and January 2017. Advanced lung adenocarcinoma patients with anaplastic lymphoma kinase (ALK) rearrangement, epidermal growth factor receptor (EGFR) mutation and both negative were included in the present study. The incidence of VTE of these patients was calculated. We then randomly selected ALK-rearrangement-positive and -negative lung adenocarcinoma tissues (n=29 and n=26, respectively) and detected TF protein expression via immunohistochemistry.ResultsAt a median follow up of 2.5 years, 5.85% (n=30/513) patients with advanced lung adenocarcinoma experienced VTE. Compared to patients with EGFR mutation (n=11/218, 5.05%) or both negative (n=13/266, 4.89%), patients with ALK-rearrangement were more likely to develop VTE (n=6/29, 20.69%; P=0.006, P=0.004; respectively). In ALK-rearrangement-positive tissues, 41.67% (n=10/24) had a high TF protein expression; the incidence was significantly higher than the TF protein expression in ALK-negative tissues (11.54%, n=3/26, P=0.015).ConclusionsALK-rearrangement-positive NSCLC patients are more likely to develop VTE; this might be due to a higher TF expression in tumor tissues.

Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells.

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.

Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes

BackgroundDiabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood.MethodsIn a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity.ResultsIn patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, d-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181−/− animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181−/− mice showed increased TF expression and FXa generation upon stimulation with LPS.ConclusionsmiR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.

Effect of blood flow on platelets, leukocytes, and extracellular vesicles in thrombosis of simulated neonatal extracorporeal circulation

BackgroundExtracorporeal membrane oxygenation (ECMO) has frequent and sometimes lethal thrombotic complications. The role that activated platelets, leukocytes, and small (0.3‐micron to 1‐micron) extracellular vesicles (EVs) play in ECMO thrombosis is not well understood. ObjectivesTo test the effect of blood flow rate on the generation of activated platelets, leukocytes, and EVs in a simulated neonatal ECMO circuit using heparinized human whole blood. MethodsSimulated neonatal roller pump circuits circulated whole blood at low, nominal, and high flow rates (0.3, 0.5, and 0.7 L/min) for 6 h. Coagulopathy was defined by thromboelastography (TEG), STA®‐procoagulant phospholipid clot time (STA®‐Procoag‐PPL), and calibrated automated thrombogram. High‐resolution flow cytometry measured the cellular expression of prothrombotic phospholipids and proteins on platelets, leukocytes, and EV. ResultsDespite heparinization, occlusive thrombosis halted flow in two of five circuits at 0.3 L/min and three of five circuits at 0.7 L/min. None of the five circuits at 0.5 L/min exhibited occlusive thrombosis. Phosphatidylserine (PS)‐positive platelets and EVs increased at all flow rates more than blood under static conditions (P < .0002). Tissue factor (TF)‐positive leukocytes and EVs increased only in low‐flow and high‐flow circuits (P < .0001). Tissue factor pathway inhibitor (TFPI), at 50 times more than the concentration in healthy adults, failed to suppress thrombin initiation in low‐flow and high‐flow circuits. ConclusionsThis in vitro study informs ECMO specialists to avoid low and high blood flow that increases TF expression on leukocytes and EVs, which likely initiate clot formation. Interventions to decrease TF generated by ECMO may be an effective approach to decrease thrombosis.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand whether polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte-derived macrophages to proinflammatory and alternative activation states. Alternative polarization with interleukin-4 and interleukin-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription- 6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased TF expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset did not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon- γ with an increase in TF production seemed to be due to an increase in TF promoter methylation and was reversible when these macrophages were treated with a demethylating agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF-bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

SYNTHETIC CANNABINOIDS: A LESS UNDERSTOOD CAUSE OF ACUTE CORONARY SYNDROME

SESSION TITLE: Monday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM PURPOSE: Current research suggests there is an association between natural cannabinoid use and acute coronary syndrome (ACS). Via an extensive literature review we will describe how synthetic marijuana can cause ACS in patients lacking typical cardiac risk factors. METHODS: Cardiology, critical care, and emergency medicine journals were searched for keywords including marijuana, tetrahydrocannabinol (THC), K2/spice in addition to ACS, myocardial infarction and sudden death. Inclusion criteria were topic relevance, peer review, and recent publication. RESULTS: A Norwegian study revealed THC as the only drug present in six postmortem patients with unexplained sudden death. Another larger study identified a 4.8-fold increase in acute myocardial infarction risk within an hour of marijuana use. Multiple case reports have linked synthetic derivatives, known as K2/spice, with ACS in young patients with no known health issues including three cases of myocardial infarction in teenage boys as well as several ACS events in healthy adults. CONCLUSIONS: An active component of both natural and synthetic marijuana is THC. It blocks parasympathetic pathways via norepinephrine release and is thought to be the cause of most cardiovascular effects. One study found a 6-53% increase in heart rate and a 4-9% rise in cardiac output after smoking marijuana, while another found a 30% increase in cardiac output and a dose-dependent increased heart rate of 20-100%. Both factors can lead to oxygen demand/delivery mismatch, which marijuana exacerbates further by increasing carboxyhemoglobin. THC can also disrupt oxygen delivery via vasospasm of the coronary vessels, increased shear stress of the arterial walls, and by forming a procoagulant state due to increased tissue factor expression in monocytic cells. Cannabinoid receptor type 1 (CB1R) is one of two receptors found abundantly in the central and peripheral nervous systems. When bound by THC, CB1R triggers autonomic responses which directly affect the vascular endothelial and smooth muscles, myocardium, and circulating erythrocytes. A recent study found CB1R leads to p38 and JNK mitogen-activated protein kinase (MAPK) activation. This increases reactive oxygen species (ROS) generation and contributes to endothelial and cardiomyocyte cell dysfunction or death. Synthetic cannabinoids are mixtures of several synthetic CB1R agonists. They can be up to 170-times more potent, leading to severe cardiac outcomes. CLINICAL IMPLICATIONS: More research is needed to fully understand effects of synthetic cannabinoids on the cardiovascular system, as synthetic marijuana may be much more dangerous than natural cannabis. It is paramount for both emergency and critical care physicians to be cognizant of the possible detrimental effects of synthetic marijuana in order to prevent ACS related events in the future. DISCLOSURES: No relevant relationships by Gary Esper, source=Web Response No relevant relationships by Peter Nesbitt, source=Web Response No relevant relationships by Ravi Patel, source=Web Response No relevant relationships by Heidi Storer, source=Web Response No relevant relationships by O'Rell Williams, source=Web Response No relevant relationships by Thijs Wolf, source=Web Response

Adipocytes express tissue factor and FVII and are procoagulant in a TF/FVIIa-dependent manner

Background: Tissue factor (TF) combined with its ligand FVII initiates blood coagulation and intracellular signaling. Obese and type 2 diabetic subjects have increased TF expression in their adipose tissue and an increased risk for thrombotic complications. Here we address the role of TF/FVII on adipocyte functions.Materials and methods: Subcutaneous fat was obtained by means of needle aspiration from healthy volunteers, and adipocytes were isolated after collagenase digestion. 3T3-L1 fibroblasts kept in culture were differentiated into adipocytes by addition of IBMX, dexamethasone, rosiglitazone, and insulin to the media. Proteins and mRNA were analyzed by western blot and RT-PCR. Coagulation activity was determined by a colorimetric FX-assay. Lipolysis was measured as free glycerol using a colorimetric method. Glucose uptake was evaluated by scintillation counting of D-[U-14C] glucose.Results: In isolated human primary adipocytes we found expression of TF and FVII. TF expression was confirmed in 3T3-L1 adipocytes, and both cell types were found to be procoagulant in a TF/FVIIa-dependent manner. FXa was generated without FVIIa added to the coagulation assay, and active site-inhibited FVIIa blocked FXa formation, supporting our finding of FVII production by human primary adipocytes. There was no evidence for a role of TF in either lipolysis or glucose uptake in our experimental settings.Conclusion: Human primary adipocytes express active TF and FVII, and the TF/FVIIa complex formed on the adipocyte surface can activate substrate FX. Whether the TF/FVIIa complex conveys signaling pathways leading to biological functions and has any biological activity in adipocytes beyond coagulation remains to be elucidated.

Evaluation of Tissue Factor -603 A/G Promoter Gene Polymorphism in Thrombotic Processes in Patients with Antiphospholipid Syndrome

<strong>Objective</strong>: Major clinical manifestations of Antiphospholipid syndrome (APS) are trombosis and thrombosis related complications. Tissue factor (TF) serves primary initiatory of the in vivo coagulation. TF gene expression may be induced in endothelial cells to initiate thrombus formation. In this study, we investigated the frequency of TF 603A/G promoter gene polymorphism in patients with antiphospholipid syndrome (APS) and its effects on TF expression and thrombotic events. <br> <strong>Materials and Methods</strong>: This study included 64 patients with APS (APS patient with thrombosis arterial and venous (n=38), antiphospholipid antibody-positive patients without thrombosis (n=26) and 44 healthy controls (HCs). Plasma TF levels and antiphospholipid antibodies were measured by ELISA method. Lupus anticoagulant was measured by activated partial thromboplastin time and kaolin clotting time. TF-603 A/G polymorphism was investigated by PCR method. <br> <strong>Results</strong>: Our results have showed that patients with APS, TF levels were found in patients significantly higher than the healthy control group (p: 0.006, p&lt;0.001, respectively). Compared between the two patient groups, TF level was significantly higher in the patient with thrombosis group compared to the patient without thrombosis group (p: 0.027). <br> <strong>Conclusion</strong>: In this study, we suggest that thrombotic complications observed in patients with APS are associated with increased TF expression.&nbsp;

Zika Virus Infection Induces Elevation of Tissue Factor Production and Apoptosis on Human Umbilical Vein Endothelial Cells.

Zika virus (ZIKV) infection is typically characterized by a mild disease presenting with fever, maculopapular rash, headache, fatigue, myalgia, and arthralgia. A recent animal study found that ZIKV-infected pregnant Ifnar−/−mice developed vascular damage in the placenta and reduced amount of fetal capillaries. Moreover, ZIKV infection causes segmental thrombosis in the umbilical cord of pregnant rhesus macaques. Furthermore, several case reports suggest that ZIKV infection cause coagulation disorders. These results suggest that ZIKV could cause an alteration in the host hemostatic response, however, the mechanism has not been investigated thus far. This paper aims to determine whether ZIKV infection on HUVECs induces apoptosis and elevation of tissue factor (TF) that leads to activation of secondary hemostasis. We infected HUVECs with two ZIKV strains and performed virus titration, immunostaining, and flow cytometry to confirm and quantify infection. We measured TF concentrations with flow cytometry and performed thrombin generation test (TGT) as a functional assay to assess secondary hemostasis. Furthermore, we determined the amount of cell death using flow cytometry. We also performed enzyme-linked immunosorbent assay (ELISA) to determine interleukin (IL)-6 and IL-8 production and conducted blocking experiments to associate these cytokines with TF expression. Both ZIKV strains infected and replicated to high titers in HUVECs. We found that infection induced elevation of TF expressions. We also showed that increased TF expression led to shortened TGT time. Moreover, the data revealed that infection induced apoptosis. In addition, there was a significant increase of IL-6 and IL-8 production in infected cells. Here we provide in vitro evidence that infection of HUVECs with ZIKV induces apoptosis and elevation of TF expression that leads to activation of secondary hemostasis.

A unique protein kinase C‐dependent pathway for tissue factor downregulation in pericytes

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11h to 4.5h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinaseC (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.