Increased Tissue Factor Expression Research Articles

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium and the modulatory mechanism of D39

Non‐muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor‐α (TNF‐α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose‐dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF‐κB p65 nuclear translocation and I B degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3 and suppressed NF‐κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF‐α‐stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model. Moreover, D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior venacava‐ligated mice dose‐dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β–NF‐κB signalling pathways. Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders. In addition, we identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.Support or Funding InformationThe present research was supported by the National Natural Science Foundation of China (Grant No. 81274131 and 81601034), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancement of Tissue Factor Expression in Monocyte-Derived Dendritic Cells by Pentraxin 3 and Its Modulation by C1 Esterase Inhibitor

Background: We have previously shown that human monocyte-derived dendritic cells (moDCs) may participate in immune system-mediated hypercoagulable state through enhanced tissue factor (TF) expression and that the complement system may be involved in this process. Objectives: The aim of this study was to explore the role of pentraxin 3 (PTX3) and the complement system in enhanced TF expression in moDCs. Methods: moDCs were generated from isolated human monocytes. PTX3 levels in whole human blood supplemented with moDCs were determined after lipopolysaccharide (LPS) stimulation. PTX3 release by the generated moDCs upon LPS stimulation was also assessed. The effect of PTX3 on whole blood coagulation was investigated using thromboelastometric analysis. TF expression in stationary moDCs treated with LPS and/or PTX3 was determined by measuring TF activity. The effect of complement inhibitors on TF activity in moDCs treated with LPS and/or PTX3 under low-shear conditions was evaluated. Results: PTX3 levels were higher in whole blood supplemented with moDCs than in the presence of monocytes and were further elevated by LPS stimulation. PTX3 release from generated moDCs was also increased by LPS stimulation. PTX3 reduced whole blood coagulation time in a dose-dependent manner. However, PTX3 did not increase TF expression in stationary moDCs. Under low-shear conditions, PTX3 increased TF expression in moDCs. C1 esterase inhibitor (C1-inh) suppressed this effect. Conclusions: PTX3 might have a thrombophilic activity and enhance TF expression in moDCs under low-shear conditions. Furthermore, suppression of moDC-associated hypercoagulability by C1-inh might be partly ascribed to its inhibitory effect on PTX3.

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles

ABSTRACTAirway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

KLF11 (Krüppel-Like Factor 11) Inhibits Arterial Thrombosis via Suppression of Tissue Factor in the Vascular Wall.

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Trimethylamine N-oxide promotes tissue factor expression and activity in vascular endothelial cells: A new link between trimethylamine N-oxide and atherosclerotic thrombosis

Trimethylamine-N-oxide (TMAO), one of the products in choline metabolite, is recently reported to be associated with cardiovascular diseases (CVD) that mainly attribute to atherothrombosis. However, the mechanisms how TMAO functions in the pathogenesis of CVD and atherothrombosis remain elusive. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. In the present study, we demonstrated that TMAO promoted TF (but not TF pathway inhibitor) expression via activation of NF-κB signaling pathway in primary human coronary artery endothelial cells (HCAECs). TMAO strongly increased TF activity and thrombin production. Further, a small dose of TMAO significantly increased TF expression and nuclear translocation of NF-κB with the synergistic action of low-dose of pro-atherosclerotic factors, such as TNF-α and HMGB1. Importantly, plasma TMAO level was positively correlated with TF activity in patients with ST-elevation myocardial infarction (STEMI). Altogether, our data revealed that TMAO promoted thrombosis through increasing TF expression and activity. The understanding of the new link between TMAO and atherothrombosis may facilitate therapeutic strategy in the prevention and treatment of atherothrombosis.

Neutrophil KLF2 Regulates Arterial and Venous Thrombosis

Experimental, clinical and pathological studies support an important link between inflammation and thrombosis. Although accumulating evidence suggests that cells of the innate immune system contribute to the thrombotic process, the identity of nodal molecular determinants operative in immune cells remains poorly understood. Our previous work in mice bearing global deletion of the transcription factor KLF2 identifiedthis factor as a critical mediator of thrombosis). Here, using cell-specific KLF2 deleted murine models (endothelial, platelet, and myeloid- deleted KLF2) we identify myeloid KLF2 as the major determinant of thrombosis. Complete blood counts and coagulation assays in myeloid KLF2 deleted mice (MY-K2-KO) were similar to those in control LysM cre mice. Using two models of vascular thrombosis (carotid artery thrombosis assay, Rose Bengal model and complete inferior vena cava ligation model of venous thrombosis) we observed a robust prothrombotic phenotype in the MY-K2-KO mice as compared to control Cre mice. As LysM Cre deletes in both neutrophils and monocyte/macrophages, we next sought to determine whether one subset was dominantly responsible for the observed phenotype. Adoptive transfer of KLF2-KO neutrophils in control mice conferred both the arterial and venous pro-thrombotic phenotype. Conversely, neutrophil depletion with Ly6G antibody (>90% depletion achieved) reversed the prothrombotic phenotype in MY-K2-KO mice. By contrast, depletion of monocytes/macrophages using clodronate liposome injection did not affect arterial or venous thrombosis. Next, we evaluated the effect of KLF2 deficiency on critical neutrophil functions that may contribute to the pro-thrombotic phenotype. As compared to controls, MY-K2-KO neutrophils demonstrated more pronounced recruitment to the endothelium. In microflow chamber assays, MY-K2-KO neutrophils showed decreased rolling and increased P-selectin induced adhesion. The importance of this was affirmed by the observation that adoptive transfer of MY-K2-KO neutrophils incubated with PSGL-1 antibody was unable to generate a prothrombotic phenotype in control mice. In addition, MY-K2-KO neutrophils demonstrated enhanced neutrophil extracellular traps generation associated with increased myeloperoxidase and neutrophil elastase expression. And finally, MY-K2-KO neutrophils (but not monocytes) demonstrated increased tissue factor expression and activity. Collectively, these studiesidentify neutrophil KLF2 as a critical determinant of both arterial and venous thrombosis and suggest that modulation of neutrophil KLF2 may be a fruitful approach to the management of thrombosis. DisclosuresSchmaier:Alnylam: Research Funding; Biomotiv: Consultancy; Cleveland Clinic Foundation: Research Funding; Temple University: Patents & Royalties; Enzyme Research Laboratories: Honoraria; Shire: Consultancy, Honoraria, Research Funding.

Venous Thromboembolism Events and Prophylaxis in Patients with Acute Myeloid Leukemia

Introduction: Thrombotic events are a frequent complication in patients with malignancy due to increased tissue factor expression and activation of hemostatic mechanisms by both host cells and cancer tissues. Patients with acute myeloid leukemia (AML) are at risk of venous thromboembolism (VTE) not only because of their malignancy but also because of prolonged hospitalizations, immobility, and the need for central venous access. Profound thrombocytopenia is an expected complication, due to myelosuppressive chemotherapy as well as from underlying marrow-infiltrative disease. This results in difficult prophylactic and treatment decisions where providers must carefully balance the risk of thrombosis and bleeding. There are no published consensus guidelines regarding appropriate use of prophylaxis in patients with leukemia.Methods: A retrospective chart review was completed on AML patients receiving induction or consolidation chemotherapy or treatment for relapsed disease admitted to the University of Virginia from October 2011 through September 2017. Patients with acute promyelocytic leukemia were excluded. Clinical data from 142 patients over a total of 667 hospital admissions were reviewed. The following data was collected: demographics (including age, sex, and race), clotting events, clot location, type of line placed, treatment of clot, and bleeding events. Bleeding events were defined according to International Thrombosis and Hemostasis criteria. χ2 analysis was performed to compare categorical values.Results: Twenty-nine of 142 (20.4%) AML patients were diagnosed with VTE. None of the patients had a prior history of clot or diagnosis of clotting disorder. Thrombosis occurred more commonly during induction therapy, 13 of the 29 events (45%), compared to other stages in their treatment (p value=0.37). Of the thromboses occurring during induction, 14 clotting events (48%) were central line associated, 4% (5/125) of thrombosis occurred in peripherally inserted central lines, 10% (5/51) in temporary internal jugular central lines and 2% (4/165) in tunneled central lines (p value=0.99).The average platelet count at the time of clot was 61 K/µl. Most patients were not on prophylaxis at the time of their clotting event (93.1% vs 6.9%; p value= <0.001). The majority of patients (19 of 29; 65.5%) were treated with therapeutic anticoagulation with heparin or low molecular weight heparin; 4 of 29 (13.8%) patients received temporary inferior vena cava filters. For those patients with a line-associated VTE, 9 of the 14 (64%) had the line removed. There was no statistically significant difference in the incidence of VTE in patient ages less than 60 years versus in older patients (55% vs 44.8%; p value=0.75).Patients who received therapeutic anticoagulation had a comparable rate of bleeding compared to patients who were not on anticoagulation (31.6% vs 40.8%; p value= 0.44). Patients who were treated for VTE (n=19) had 2 major bleeds and 4 clinically relevant non-major bleeds. Patients who did not receive treatment for their VTE event (n=10) had 1 major bleed and 3 minor bleeding events.Conclusion: Our study identified a 20.4% incidence of VTE in AML patients, occurring more frequently during induction chemotherapy and in patients without prior thrombotic history.The clots were most commonly catheter associated and the highest rate of clot occurred with temporary internal jugular lines. Importantly, we found that when patients were treated with anticoagulation, their incidence of bleed did not exceed the general incidence of bleeding (43%) seen during the time period of the analysis. Average platelet count at VTE diagnosis was 61 K/µL, which is above most institutions' thresholds for use of pharmacologic VTE prophylaxis including patients with acute leukemia. Given that only two patients in our study were receiving prophylaxis at the time of VTE and that bleeding rates were similar with full therapeutic anticoagulation, this suggests prophylaxis is important, safe, and is potentially neglected in patients with acute leukemia. This highlights an area of potential improvement in decreasing VTE in patients that can safely receive prophylaxis and is the basis for future prospective safety trials exploring the use of prophylaxis in this patient population. DisclosuresNo relevant conflicts of interest to declare.

Clinical Cellular Therapeutics Accelerate Clot Formation.

Clinical cellular therapeutics (CCTs) have shown preliminary efficacy in reducing inflammation after trauma, preserving cardiac function after myocardial infarction, and improving functional recovery after stroke. However, most clinically available cell lines express tissue factor (TF) which stimulates coagulation. We sought to define the degree of procoagulant activity of CCTs as related to TF expression. CCT samples from bone marrow, adipose, amniotic fluid, umbilical cord, multi‐potent adult progenitor cell donors, and bone marrow mononuclear cells were tested. TF expression and phenotype were quantified using flow cytometry. Procoagulant activity of the CCTs was measured in vitro with thromboelastography and calibrated thrombogram. Fluorescence‐activated cell sorting (FACS) separated samples into high‐ and low‐TF expressing populations to isolate the contribution of TF to coagulation. A TF neutralizing antibody was incubated with samples to demonstrate loss of procoagulant function. All CCTs tested expressed procoagulant activity that correlated with expression of tissue factor. Time to clot and thrombin formation decreased with increasing TF expression. High‐TF expressing cells decreased clotting time more than low‐TF expressing cells when isolated from a single donor using FACS. A TF neutralizing antibody restored clotting time to control values in some, but not all, CCT samples. CCTs demonstrate wide variability in procoagulant activity related to TF expression. Time to clot and thrombin formation decreases as TF load increases and this procoagulant effect is neutralized by a TF blocking antibody. Clinical trials using CCTs are in progress and TF expression may emerge as a safety release criterion. Stem Cells Translational Medicine 2018;7:731–739

Hemostasis at Extremes of Body Weight.

Extremes of body weight are not uncommon in the modern world and include anorexia nervosa (AN) and obesity. Both conditions are associated with increased morbidity and mortality: AN has the highest mortality rate of all mental illnesses and unfortunately obesity has reached epidemic proportions and is a well-recognized risk factor for cardiovascular disease including venous thromboembolism (VTE). This article summarizes the current understanding of hemostatic changes of these extremes of body weight. The hemostatic changes of AN have not been well described. Severe AN is associated with pancytopenia with decreased bone marrow cellularity, which causes a mild thrombocytopenia. Platelet hyperaggregability has been recognized in AN and has been attributed at least in part to increased adrenoceptor density. Obesity and the metabolic syndrome are associated with prothrombotic changes, which have been well characterized and related to complex adipocyte-induced inflammatory changes, including increased levels of plasminogen activator inhibitor type 1, von Willebrand factor, fibrinogen, and other evidence of increased coagulation and platelet activation. Accumulating evidence suggests a significant role for increased tissue factor expression and signaling in this relationship, with increased tissue factor expression present in adipose and possibly systemic tissues, induced by adipose-generated cytokines. Intriguingly, the hemostatic changes do not seem to increase with increasing BMI, although the risk of VTE increases with BMI, suggesting that decreased venous flow due to venous enlargement may play the most important role in increased VTE risk with obesity.

Experimental hypercoagulable state induced by tissue factor expression in monocyte-derived dendritic cells and its modulation by C1 inhibitor.

The crosstalk between immune and coagulation systems plays pivotal roles in host defense, which may involve monocyte-derived dendritic cells (moDCs). Our objectives were to elucidate the role of moDCs in coagulation under inflammatory conditions and the involvement of the complement system. We assessed the effects of lipopolysaccharide (LPS)-stimulated moDCs on coagulation using whole blood thromboelastometry in the presence of complement inhibitors. The sum of clotting time and clot formation time (CT plus CFT) in whole blood thromboelastometry was significantly more reduced in the presence of moDCs than in the absence of monocytes or moDCs and in the presence of monocytes, indicating a more potent coagulability of moDCs. The mRNA expression of coagulation-related proteins in moDCs was analyzed by quantitative PCR, which showed an increase only in the mRNA levels of tissue factor (TF). TF protein expression was assessed by western blot analysis and an activity assay, revealing higher TF expression in moDCs than that in monocytes. The in vitro moDC-associated hypercoagulable state was suppressed by a TF-neutralizing antibody, whereas LPS enhanced the in vitro hypercoagulation further. C1 inhibitor suppressed the in vitro LPS-enhanced whole blood hypercoagulability in the presence of moDCs and the increased TF expression in moDCs. These results suggest a significant role of moDCs and the complement system through TF expression in a hypercoagulable state under inflammatory conditions and demonstrate the suppressive effects of C1 inhibitor on moDC-associated hypercoagulation.

Abstract 158: Indoleamine 2,3-dioxygenase 1 is Expressed in Macrophages in Human Coronary Atherosclerotic Plaque and is Associated With Tissue Factor Expression in Activated THP-1 Macrophages

Background: Recently, several clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme of the Kyn pathway and is induced by cytokines, particularly IFNγ. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Objective: The present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP) and define its role in plaque thrombogenicity. Methods: Immunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with SAP and UAP. We examined the role of IDO1 in tissue factor (TF) expression in THP-1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α. Results: We localized IDO1 mainly in CD68-positive macrophages in atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3-positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. THP-1 macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF-κB (p65) binding activity in activated THP-1 macrophages. Inhibition of the aryl hydrocarbon receptor (AHR) that binds to Kyn, also reduced Kyn-induced TF expression in activated THP-1 macrophages. Kynurenine dose-dependently increased TF expression under IDO1 inhibition whereas hydroxyanthranilic and quinolinic acids did not. Conclusion: The IDO1 is expressed in coronary atherosclerotic plaques and might contribute to thrombus formation through TF upregulation via NF-κB (p65) and AHR in activated macrophages.

Androgen receptor dampens tissue factor expression via nuclear factor‐κB and early growth response protein 1

Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients.We characterized androgen receptor‐mediated tissue factor regulation in prostate epithelial cells.Androgen receptor is dampening tissue factor expression in prostate epithelial cells.Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates. SummaryBackgroundProstate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)‐mediated TF gene expression.ObjectivesTo characterize AR‐mediated TF regulation in vitro and in vivo.MethodsWe used the androgen‐dependent prostate cancer cell lines LNCaP and MyC‐CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor‐binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR‐mediated TF regulation in vivo.Results TF is directly regulated by AR. In LNCaP cells, nuclear factor‐κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells.Conclusion AR is crucial for dampening TF expression, which could be important for increased TF expression and TF‐positive microvesicle release in androgen‐deprived prostate cancer patients.

Hypercoagulable state in sickle cell disease.

Chronic activation of coagulation is one of the features of sickle cell disease (SCD). Increased tissue factor expression, phosphatidylserine exposure, thrombin generation and fibrinolysis, as well as decreased levels of natural anticoagulants have been reported in SCD patients and in the mouse models of SCD. Consistent with this, patients with SCD are prone to develop thrombotic complications. In addition, the altered morphology of sickle red blood cells (RBC) may also alter the properties and dynamics of clot formation in SCD patients. Clinical data and results from animal models have revealed complex interactions between coagulation, chronic hemolysis, and inflammation suggesting that activation of coagulation may contribute to the pathophysiology of SCD.

Involvement of avß3 integrin and PAR signaling in thrombin generation and proliferation of vascular smooth muscle cells from thoracic and abdominal aortic aneurysms

In contrast to abdominal aortic aneurysm (AAA), aneurysms of the thoracic ascending aorta (TAA) are not associated with a mural thrombus. However, we have previously shown the presence of prothrombin in the media of TAA. To determine (i) whether thrombin generation (TG) at the surface of VSMCs is increased in AAA, (ii) its dependence with respect to the etiology of the aneurysm, and (iii) the contribution of avß3 integrins as a receptor for prothrombin (FII) and PAR activation to TG and thrombin-induced proliferation. Primary cultures of VSMCs were prepared from human biopsies of TAA, AAA and healthy aorta ( n = 11/group). TG was monitored by thrombography in the presence of healthy plasma or plasma deficient in FII, factor X or VII. In the presence of prothrombin deficient plasma, similar low levels of thrombin (about 1 nM) are formed on the 3 types of VSMCs indicating the presence of FII within the media confirmed by western blot. There is no TG in plasma deficient for factor VII or X. There was no difference in avß3 integrin expression between the different VSMCs. In the presence of healthy plasma, TG was significantly higher (30% increase) on VSMCs from AAA (accompanied by an increase in tissue factor expression) and lower on VSMCs from TAA compared with healthy VSMCs. Inhibition of TG by an antagonist of PAR-1 was more pronounced in VSMCs from AAA than from TAA a without difference in PAR-1 expression. This effect was associated with increased thrombin-induced proliferation of VSMCs from AAA compared with healthy VSMCs. The higher prothrombotic effect of VSMCs from AAA is mediated partly by PAR in an auto-amplification loop and suggests the contribution of these cells in the occurrence of thrombotic events. Our results demonstrate also that endogenous synthesis of prothrombin is sufficient to produce nanomolar amounts of thrombin known to exert cell proliferative effect involved in both AAA and TAA progression.

CD39 and CD73 activity are protective in a mouse model of antiphospholipid antibody-induced miscarriages

ObjectiveAntiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS. MethodsWe studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity. ResultsaPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression. ConclusionWe demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

456P - ALK-rearranged may promote VTE by increasing the expression tissue factor (TF) in lung adenocarcinoma

Background: Patients with lung cancer are at increased risk for venous thromboembolism (VTE). About 8-15% of patients with advanced non-small-cell lung cancer (NSCLC) experience a VTE in the course of their disease. However, the incidences of VTE in different molecular subtypes of NSCLC are rarely reported though they have big differentiation in clinical features and therapeutic effects. Tissue factor (TF) expressed in many solid tumors could bind and activate coagulation factor FVII and trigger the downstream coagulation cascade leading to thrombin generation and clot formation. Methods: Here we extracted retrospective data from electronic medical records at Henan Cancer Hospital in China between January 2015 and January 2016. Lung adenocarcinomas with ALK-rearranged, EGFR mutation and both-negative were classified. The incidence rate of VTE after diagnosed with lung adenocarcinoma was calculated and analyzed. Then we randomly selected ALK-rearranged and ALK-rearranged-negative lung adenocarcinoma tissues and detected TF expression in them with imunohistochemistry. Results: At a median follow-up of 19 months, 5.85% (30 in 513) patients with advanced lung adenocarcinoma experienced VTE. Patients with different molecular subtypes showeddifferent rates of VTE (P = 0.0021). Among them ALK-rearranged were more likely to experience VTE (6 in 29, 20.69%). EGFR and both-negative had lower rates of VTE and had no big difference between them (11 in 218, 5.05%; 13 in 266, 4.89%, respectively). The expression of TF performed similar feature. TF high expression in ALK-rearranged tissues is 41.67% (10 in 24), dramatically higher than that in ALK-rearranged negative tissues (11.54%, 3 in 26, P = 0.0152). Conclusions: The rate of VTE in the ALK-rearranged advanced lung adenocarcinoma cohort was about 4-fold higher than that in EGFR mutation and both-negative patients. ALK-rearranged may promote VTE by increasing TF expression. The mechanism warrants further research. Legal entity responsible for the study: The Affiliated Cancer Hospital of Zhengzhou University Funding: None Disclosure: All authors have declared no conflicts of interest.

Myeloid-related protein-8/14 in acute coronary syndrome

BackgroundThe alarmin family member myeloid-related protein (MRP)-14 (S100A9), which has been identified by platelet transcriptional profiling as an acute myocardial infarction gene, regulates vascular inflammation and thrombosis. Elevated plasma levels of MRP-8/14 (S100A8/A9) heterodimer predict first and recurrent cardiovascular events. The aim of this study was to elucidate pathophysiological roles of MRP-8/14 in acute coronary syndrome (ACS). Methods and resultsIn 38 consecutive ACS patients, the MRP-8/14 level in coronary artery blood obtained at thrombus aspiration was higher in 23 patients, in whom aspirated thrombus was confirmed, compared to the 15 patients, in whom it was absent [4.86 (1.95, 8.29) vs 2.94 (1.31, 4.44), P=0.017]. The MRP-8/14 level was correlated with myeloperoxidase (MPO) level (R2=0.52), but not with soluble P-selectin level (R2=0.0002) in the coronary artery blood. Immunohistochemistry of the aspirated thrombus exhibited that expression of MRP8/14 was co-localized with leukocytes positive for activated Mac-1. Finally, in cultured human umbilical vein endothelial cells, MRP-8/14 increased tissue factor expression. ConclusionsOur findings indicate that MRP-8/14 concentration increases in coronary artery blood in association with thrombus formation in ACS, co-localizes with leukocytes, and is associated with leukocyte activation. MRP-8/14 is positioned as a unique biomarker at the interface of inflammation and thrombosis in ACS.

Heterodimers of the transcriptional factors NFATc3 and FosB mediate tissue factor expression for 15(S)-hydroxyeicosatetraenoic acid–induced monocyte trafficking

Tissue factor (TF) is expressed in vascular and nonvascular tissues and functions in several pathways, including embryonic development, inflammation, and cell migration. Many risk factors for atherosclerosis, including hypertension, diabetes, obesity, and smoking, increase TF expression. To better understand the TF-related mechanisms in atherosclerosis, here we investigated the role of 12/15-lipoxygenase (12/15-LOX) in TF expression. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major product of human 15-LOXs 1 and 2, induced TF expression and activity in a time-dependent manner in the human monocytic cell line THP1. Moreover, TF suppression with neutralizing antibodies blocked 15(S)-HETE-induced monocyte migration. We also found that NADPH- and xanthine oxidase-dependent reactive oxygen species (ROS) production, calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation, and interactions between nuclear factor of activated T cells 3 (NFATc3) and FosB proto-oncogene, AP-1 transcription factor subunit (FosB) are involved in 15(S)-HETE-induced TF expression. Interestingly, NFATc3 first induced the expression of its interaction partner FosB before forming the heterodimeric NFATc3-FosB transcription factor complex, which bound the proximal AP-1 site in the TF gene promoter and activated TF expression. We also observed that macrophages from 12/15-LOX-/- mice exhibit diminished migratory response to monocyte chemotactic protein 1 (MCP-1) and lipopolysaccharide compared with WT mouse macrophages. Similarly, compared with WT macrophages, monocytes from 12/15-LOX-/- mice displayed diminished trafficking, which was rescued by prior treatment with 12(S)-HETE, in a peritonitis model. These observations indicate that 15(S)-HETE-induced monocyte/macrophage migration and trafficking require ROS-mediated CaMKIV activation leading to formation of NFATc3 and FosB heterodimer, which binds and activates the TF promoter.

Increased tissue factor expression and promoter hypomethylation in preeclampsia placentas in a Chinese population.

To explore the relationship between expression of tissue factor and its methylation levels in the placentas of preeclampsia among Chinese Han population. In this study, we explored gene manifestation or expression of tissue factor, and evaluated methylation levels of tissue factor promoter areas in 16 clinical preeclampsia placentas as well as 16 paired normal pregnancy placentas from the Chinese Han population. Real-time PCR, immunohistochemistry, Western blotting, and methylation-specific PCR were conducted. Expression of tissue factor protein in the preeclampsia group was 0.857±0.043, which was considerably elevated over that in the control group (0.248±0.035) (P<0.05). Expression of tissue factor was elevated in mRNA as well as in protein quantities in preeclampsia placentas, compared with the control group (1.45±0.42vs. 0.25±0.28, P<0.05). Immunohistochemical staining for tissue factor showed positive within syncytio-cytotrophoblast layers, and there was additional scattered staining in the preeclampsia group. Methylation-specific PCR showed that methylation levels of tissue factor promoter were considerably reduced in preeclamptic placentas (0.928±0.148), in comparison with control group (0.187±0.112, P<0.05). Tissue factor is expected to participate in preeclampsia etiology through methylation regulation.

Senescence of Pancreas in Middle-Aged Rats with Normal Vascular Function.

BACKGROUND In organ transplantation, particularly pancreas transplantation, donor age is a determinant factor for graft survival. Physiological aging is crucial in the progressive deterioration of organs in adulthood. We compared the senescence and function features of pancreas and vascular tissues in young rats and middle-aged rats. MATERIAL AND METHODS Islet morphology and the area of cells secreting insulin or glucagon was investigated using immunohistology in young rats (12 weeks) and middle-aged rats (52 weeks) (n=8). Senescence markers, oxidative stress (ROS), and tissue factor (TF) were measured in the rat pancreases. Circulating microparticles (MPs) were measured as surrogates of vascular cell injury. Vascular function was studied in mesenteric arterial rings. RESULTS Larger islets were twice as frequent in young rats versus middle-aged rats. In middle-aged rats there was a significant decrease of the β-cells/islet area ratio. Western blot analysis showed an increased expression of p53, p21, and p16 senescence markers (2-, 7- and 3-fold respectively) with no modification in caspase-3 activation. A 30% decrease of endothelial nitric oxide synthase (eNOS) was observed together with a 4-fold increase in TF expression. ROS formation increased significantly (2-fold) in middle-aged rats and their main source, determined by pharmacological inhibition, was NADPH oxidase and uncoupled nitric-oxide (NO) synthase. No sign of vascular injury (microparticles) or dysfunction was evidenced. CONCLUSIONS Modification in islet morphology and function were detected in middle-aged rats before any measurement of macro-vascular dysfunction. The data indicate a pancreatic senescence in the process of aging associated with uncontrolled accumulation of oxidative species that suggests a determining role of donor age in transplantation.