Tissue Factor Procoagulant Activity Research Articles

OC-09 - Microparticle-mediated transfer of TF hypercoagulability between cancer cells

Tissue Factor (TF) promotes apoptosis-resistance and facilitates haematogenous metastasis. Cancer cells release TF-bearing microparticles (TF-MP) which have pro-coagulant activity. TF-MPs from cancer cells induce increase in TF expression and procoagulant activity of recipient endothelial cells. Drug resistance microparticles transfer between cancer cells, resulting in induction of recipient cell drug resistance, however transfer of TF-MPs between cancer cells has not been demonstrated. To determine if microparticles isolated from TF expressing cancer cell lines can induced TF-mediated procoagulant activity in cell lines that do not express TF. A7 cells (a melanoma cell line that does not express TF) were transformed to express TF with adenoviral transformation. In addition, the TF-expressing breast cancer cell line MDAMB231 was used. Media was collected from (1) wild-type A7 (A7-WT) cells, which acted as control (2) MDAMB231 cells, (3) A7 cells transformed with 'low' levels of TF-adenovirus and (4) A7 cells transformed with 'high' levels of TF-adenovirus. MPs were isolated from media by centrifugation using standard techniques. Isolated MPs were incubated with wild-type A7 (A7-WT) cells overnight. Pro-coagulant activity of A7-WT cells was assessed using a FXa generation assay. i) A7-WT cells incubated overnight with MDAMB231-derived MPs produced an almost 5 fold increase in procoagulant activity compared to control (Mean 3.5 mOD/min S.E 0.32 vs control 0.68 S.E. 0.05, p<0.01). ii) MPs released from A7-TF cells induced a dose-dependant increase in cell based procoagulant activity when incubated with A7-WTs compared to control:A7-WT cells incubated with control MPs (Control): mean mOD 0.83, S.E. 0.07A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 1.27, S.E. 0.06 (p=0.04 vs control)A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 2.77, S.E. 0.23 (p<0.01 vs control). These results demonstrate, for the first time, microparticle-mediated transfer of TF procoagulant activity between cancer cells. MPs shed by cancer cells in vivo have been implicated in cancer cell progression and hypercoagulability. The acquisition and spread of of MP-mediated TF activity has implications for cancer cell biology and cancer-induced hypercoagulability.

Pregnancy-associated plasma protein-A promotes TF procoagulant activity in human endothelial cells by Akt-NF-κB axis.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a controversial role in pathophysiology of cardiovascular disease. It seems involved in progression of atherosclerosis and is widely represented in atherosclerotic plaque. PAPP-A plasma levels are elevated in patients with acute coronary syndromes (ACS), thus it has been suggested that it might be a prognostic marker for developing major cardiovascular events. However, the pathophysiological link(s) between PAPP-A and ACS are still unknown. Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates whether PAPP-A, at concentrations measurable in ACS patients, might induce TF expression in human endothelial cells in culture (HUVEC). In HUVEC, PAPP-A induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by FACS analysis and pro-coagulant activity assay. PAPP-A induced TF expression through the activation of Akt/NF-κB axis, as demonstrated by luciferase assay and by suppression of TF-mRNA transcription as well as of TF expression/activity by Akt and NF-κB inhibitors. These data indicate that PAPP-A promotes TF expression in human endothelial cells and support the hypothesis that this proteinase, besides being involved in progression of atherosclerosis, does not represent an independent risk factor for adverse cardiovascular events, but it rather might play an "active" role in the pathophysiology of ACS as an effector molecule able to induce a pro-thrombotic phenotype in endothelial cells.

Microvesicle-associated tissue factor procoagulant activity for the preoperative diagnosis of ovarian cancer

BackgroundTissue factor (TF) is involved in tumor growth and metastasis and contributes to venous thromboembolism (VTE) in cancer, including gynecological malignancies. The diagnostic value of microvesicle-associated TF procoagulant activity (MV TF PCA) in women with suspected ovarian cancer, however, has not been studied. ObjectiveTo evaluate MV TF PCA as a diagnostic tool in women with an ovarian mass of unknown etiology and as a predictive biomarker for perioperative VTE. MethodsPlasma MVs were isolated by high-speed centrifugation and analyzed for TF-specific PCA by single-stage clotting assay. In addition, plasma TF antigen and soluble P-selectin (sCD62P) were measured by ELISA. ResultsD-Dimer, MV TF PCA, and sCD62P, but not the tumor marker, CA-125, significantly differentiated patients with malignant (n=40) from those with benign tumors (n=15) and healthy controls (n=34). In cancer patients, only D-Dimer and CA-125 correlated with the FIGO stage. An abnormal D-dimer had the highest sensitivity for the diagnosis of cancer, while MV TF PCA above the ROC curve-derived cut-off value of 182U/mL had the highest specificity. By multivariate logistic regression analysis, addition of MV TF PCA conferred diagnostic benefit to the single variables, CA-125 (p=0.052) and D-dimer (p=0.019). Perioperative VTE occurred in 16% of cancer patients and was associated with an advanced FIGO stage, but not MV TF PCA. There was no difference in plasma TF antigen levels between study groups. ConclusionsMV TF PCA, but not plasma TF antigen, may provide valuable additional information for the diagnostic work-up of women with suspected ovarian cancer.

Effect of anti-GRP78 autoantibodies on prostate cancer progression through tissue factor procoagulant activity.

258 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increase PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumour growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titres were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumour growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs.

Tissue Factor Regulation by miR-520g in Primitive Neuronal Brain Tumor Cells: A Possible Link between Oncomirs and the Vascular Tumor Microenvironment

Pediatric embryonal brain tumors with multilayered rosettes demonstrate a unique oncogenic amplification of the chromosome 19 miRNA cluster, C19MC. Because oncogenic lesions often cause deregulation of vascular effectors, including procoagulant tissue factor (TF), this study explores whether there is a link between C19MC oncogenic miRNAs (oncomirs) and the coagulant properties of cancer cells, a question previously not studied. In a pediatric embryonal brain tumor tissue microarray, we observed an association between C19MC amplification and reduced fibrin content and TF expression, indicative of reduced procoagulant activity. In medulloblastoma cell lines (DAOY and UW228) engineered to express miR-520g, a biologically active constituent of the C19MC cluster, we observed reduced TF expression, procoagulant and TF signaling activities (responses to factor VIIa stimulation), and diminished TF emission as cargo of extracellular vesicles. Antimir and luciferase reporter assays revealed a specific and direct effect of miR-520g on the TF 3' untranslated region. Although the endogenous MIR520G locus is methylated in differentiated cells, exposure of DAOY cells to 5-aza-2'-deoxycytidine or their growth as stem cell-like spheres up-regulated endogenous miR-520g with a coincident reduction in TF expression. We propose that the properties of tumors harboring oncomirs may include unique alterations of the vascular microenvironment, including deregulation of TF, with a possible impact on the biology, therapy, and hemostatic adverse effects of both disease progression and treatment.

Tissue Factor Decryption By Oxidative Stress-Induced Lipid Peroxidation: Potential Mechanisms

Cellular lipid peroxidation is known to contribute to the initiation and propagation of atherothrombosis. Recently, we showed that 4-hydroxynonenal (HNE), one of the most abundant reactive aldehydes generated from the oxidation of ω-6 fatty acids, enhanced tissue factor (TF) activity on monocytic cells by externalizing phosphatidylserine (PS) in p38 MAPK activation-dependent manner. However, at present, the link between HNE-induced oxidative stress and p38 MAPK activation and the relation of p38 MAPK activation to PS externalization is not fully known. In the present study, we investigated the role of mitochondrial electron transport chain and reactive oxygen species (ROS) generation in HNE-mediated TF decryption. In addition, we also investigated the thioredoxin reductase-thioredoxin-ASK-1 axis in regulating p38 MAPK activation and PS externalization in decrypting TF. To elucidate potential mechanisms of HNE-induced TF decryption, we first determined the role of specific mitochondrial electron transport chain complexes in regulating TF activity. Since THP-1 cells used in the study had a measurable basal TF activity, they were not further treated with LPS or other agonists to induce TF synthesis. The electron transport chain in these cells was disrupted by specific inhibitors and cell surface TF activity was measured by factor X activation assay. Inhibition of complex I and complex IV by rotenone and sodium azide, respectively, enhanced the procoagulant activity of basal level TF. However, the inhibition of complex I and IV had no significant effect on the HNE-mediated increase in TF activity. Interestingly, inhibition of ATP synthase/complex V by oligomycin significantly inhibited the HNE-mediated enhanced TF activity, indicating that HNE-mediated TF decryption may involve the generation of ATP. In agreement with earlier published studies in monocytes/macrophages, stimulation of THP-1 cells with ATP increased cell surface TF activity. However, at present, it is yet to be shown that HNE treatment actually increased the production of ATP and that this ATP is responsible for the HNE-mediated TF decryption. It is also possible that HNE, either through a generation of ROS in mitochondria or directly, can affect the activity of thioredoxin either by intracellular signaling or by directly forming an adduct with it. Therefore, we next investigated the effect of HNE on the activity of thioredoxin reductase, the enzyme known to regulate thioredoxin activity in the cell. Our data showed that HNE treatment inhibited the activity of thioredoxin reductase in a concentration-dependent manner, 40 µM of HNE inhibiting 50% of the activity and a complete inhibition at 80µM of HNE. To further determine the downstream signaling cascade involved in the PS externalization and TF decryption on exposure to HNE, we analyzed the effect of HNE on the activation of MKK3 and MKK6, the protein kinases known to activate p38 MAPK and the downstream signaling activator of thioredoxin/thioredoxin reductase pathway. HNE treatment increased the phosphorylation of MKK3 and MKK6 in a time-dependent manner. In summary, our data suggest that HNE may mediate TF decryption via modulation of thioredoxin/thioredoxin reductase system, which results in activation of MKK3/MKK6, which in turn activates p38 MAPK that is responsible for PS externalization. The study highlights the potential role of oxidative stress in regulating TF activity in thrombotic disorders and provides a mechanistic link between disorders associated with cellular oxidative stress and thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Hyperactivatable Protein C Expression Partially Rescues the Embryonic Lethality of TFPI Null Mice but Surviving Mice Develop Severe Hydrocephalus

Introduction: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two anticoagulant proteins directly bound to endothelium. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and early forms of prothrombinase. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. An in vitro model system of purified coagulation proteins has shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. We have previously shown that mice with decreased thrombomodulin function (TMpro) do not have cerebral fibrin deposition even following LPS injection, while partial TFPI deficiency does induce intravascular fibrin deposition in the brain of the TMpro mice, suggesting that these two anticoagulant proteins have distinct functions within the brain vasculature. TFPI null mice die in utero with brain fibrin deposition. A hyperactivatable murine protein C (hMPC) can be produced by altering two acidic amino acids near the thrombin cleavage site. hMPC is activated 30-fold more efficiently by thrombin than wild type protein C and its activation does not require TM. We have used murine model systems to investigate how hMPC expression alters the (patho)physiology of mice with TFPI deficiency.Methods: Transgenic mice producing hMPC under control of the transthyretin promoter were produced. hMPC was bred into TFPI+/- mice, which were then characterized to define the anticoagulant activity of the transgene. Offspring from these initial matings were interbred to determine if hMPC expression would rescue the embryonic lethality of TFPI null mice.Results: hMPC expression elevated plasma protein C 2-fold and plasma APC 3-fold producing a potent anticoagulant effect. In thrombin generation assays, plasma from TFPI+/-/hMPC+ mice had peak thrombin generation of 44.9±7.5 nM vs. 63.0±3.5 nM in TFPI+/-/hMPC- mice (p<0.001). In a TF-induced pulmonary embolism model 5 of 14 TFPI+/-/hMPC+ mice survived over 5 minutes, while only 1 of 10 TFPI+/-/hMPC- did (p=0.02). When TFPI+/-/hMPC+ mice were mated with TFPI+/-/hMPC- mice, TFPI-/-/hMPC+ pups were born at approximately 30% of the expected frequency regardless of whether the transgene was expressed by the mother or the father. About 35% of the surviving TFPI-/-/hMPC+ mice developed a domed shaped head and succumbed to severe hydrocephalus by 8 weeks of age. Those surviving beyond 8 weeks did not develop severe hydrocephalus and were fertile. One TFPI-/-/hMPC+ mouse with a domed shaped head survived long enough to be examined by MRI, which documented severe hydrocephalus. In addition, two lesions (1mm and 0.5 mm diameter) were present in remaining brain tissue. These lesions contained iron suggesting they were areas of blood clot or hemorrhage. MRI exam of 12 week old TFPI-/-/hMPC+ mice identified subclinical hydrocephalus in 1 of 4 mice. Hydrocephalus did not occur in TFPI+/+/hMPC+ or TFPI+/-/hMPC+ mice. Histological examination of brain tissue from TFPI-/-/hMPC+ mice confirmed hydrocephalus with little remaining normal brain tissue. Mice with hydrocephalus had hemorrhage in the ventricles and brain parenchyma that was associated with areas of fibrin(ogen) deposition. There was also congestion of pial vessels and hemorrhage within the subarachnoid space. These findings were not observed in TFPI+/-/hMPC+ mice.Conclusions: Expression of the hMPC transgene produces a potent anticoagulant effect that partially rescues TFPI null embryonic lethality. Surviving TFPI-/-/hMPC+ pups are susceptible to death from severe hydrocephalus associated with hemorrhage and vascular abnormalities in the brain as they mature. These findings demonstrate that TFPI produces physiologically important anticoagulant activity within murine brain vasculature that is not fully compensated by over-expression of activated protein C. DisclosuresMast:Novo Nordisk: Honoraria, Research Funding.

Tissue Factor Pathway Inhibitor (TFPI) May be Another Important Factor in the Coagulopathy in Acute Promyelocytic Leukemia (APL)

The characteristic coagulopathy in Acute Promyelocytic Leukemia (APL) is unique among the leukemias and thrombotic and bleeding complications remain the major causes of early deaths. APL promyelocytes express tissue factor (TF) which, after activation by phospholipids, forms a complex with factor VII and converts factor X to activated factor X. TF plays a central role in the pathogenesis of APL coagulopathy, for the procoagulant activity of lysates from freshly isolated APL cells is mainly attributed to TF. During periods of increased apoptosis of promyelocytes, the procoagulant activity is correspondingly intensified. In addition, APL cells may also induce TF procoagulant activity of endothelial cells through their secretion of IL-1b. The activity of the complex FVIIa-TF can be inhibited by the tissue factor pathway inhibitor (TFPI), but its role in the APL-associated coagulopathy remains unknown. This study aimed to determine the time-course of TFPI levels in patients with APL at diagnosis and during the first two-weeks of treatment with ATRA and anthracyclines. Twenty patients with de novo APL (12 males, age ranging from 17 to 72 years) and 20 healthy blood donors (age and sex matched) were included in this study. Peripheral blood (PB) samples were collected at diagnosis, and at D3, D7 and D15 of the induction therapy with ATRA and daunorubicin. The following plasma concentrations were determined: Thrombin-Antithrombin complex (TAT) (Enzygnost¨ TAT micro, Dade Behring/Siemens, IL, EUA), total TFPI (Asserachrom¨ total TFPI, Diagnostica Stago, Frana) and free TFPI (Asserachrom¨ free TFPI, Diagnostica Stago, Frana). The plasma concentrations of TAT were significantly higher in APL patients at presentation, D3 and D7 compared to controls, suggesting that the pathologic activation of coagulation was reversed by D15 of treatment Figure 1). The concentrations of free-TFPI were significantly higher in APL samples compared to controls in all time-points (Figure 2). In contrast, the concentrations of the truncated form of TFPI (estimated by subtracting the free-TFPI values from total-TFPI concentration) did not vary significantly between the groups. Since, free-TFPI (intact form) has been proven to present higher capacity to inhibit the activated factor X and VIIa-TF proteases than truncated-TFPI (bound to lipoprotein), we hypothesized that our findings confirm the relevance of free-TFPI in APL coagulopathy. To test whether the administration of rh-TFPI may revert aberrant activation of coagulation in APL patients, we developed a murine model using the IV infusion of NB4 cells. Similarly to the reported in APL patients, we observed an increase on TAT levels in mice injected with 5x106 NB4 cells [(60.7 ng/ml (24 - 116 ng/ml) vs 11.5 ng/ml (2.2 - 27.8 ng/ml), p<0.05)]. In contrast, murine TFPI levels decreased 30 minutes after the infusion of NB4 cells [30 ng/ml (5.8 - 217 ng/ml) vs 202 ng/ml (46 - 354 ng/ml)], suggesting that it was rapidly consumed. Unfortunately, it was not possible to distinguish between truncated and intact TFPI forms in mice. Intravenous infusion of recombinant human (rh) TFPI at the dose of 10mg/Kg significantly decreased the TAT levels (Figure 3) and increased TFPI levels in the murine model. In conclusion, our data on clinical samples and pre-clinical data using a murine model suggest that treatment with rh-TFPI may be a strategy to counteract APL-associated coagulopathy. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tissue factor-dependent and -independent pathways of systemic coagulation activation in acute myeloid leukemia: a single-center cohort study.

BackgroundIn acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood.MethodsWe conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye.ResultTF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality.ConclusionIn newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy

BackgroundPatients with germ cell tumors (GCTs) receiving cisplatin-based chemotherapy are at increased risk of thrombosis, but the underlying cellular and molecular mechanisms remain obscure. ObjectiveTo study baseline tissue factor (TF) expression by GCT cell lines and its modulation by cisplatin treatment. MethodsTF expression was assessed by single-stage clotting and thrombin generation assay, flow cytometry, ELISA, and Western blot analysis. Cell cycle analysis and detection of phosphatidylserine (PS) membrane exposure were carried out by flow cytometry. TF mRNA was analyzed by quantitative RT-PCR. ResultsSignificant expression of TF-specific procoagulant activity (PCA) was detected on three non-seminoma (NT2, 2102Ep, NCCIT) and one seminoma cell line (TCam-2). Treatment with 0.4μM cisplatin (corresponding to the IC50) for 48hrs increased TF PCA on NT2 cells 3-fold, an effect that was largely independent of PS exposure and that could not be explained by translocation of active TF from intracellular storage pools. Cisplatin-induced TF PCA expression in NT2 cells did not occur before 12hrs, but was steady thereafter and accompanied by a 2-fold increase in total and surface-located TF antigen. Importantly, increased TF gene transcription or production and release of an intermediate factor were not involved in this process. Cell cycle analysis suggested that cisplatin-induced G2/M arrest resulted in an accumulation of procoagulant TF on the membrane surface of NT2 cells. ConclusionsIn addition to induction of apoptosis/necrosis with PS-mediated activation of preformed TF, cisplatin may alter the procoagulant phenotype of GCT cells through an increase in total cellular TF antigen.

Tissue factor and Toll-like receptor (TLR)4 in hyperglycaemia-hyperinsulinaemia. Effects in healthy subjects, and type 1 and type 2 diabetes mellitus.

Diabetes mellitus (DM) patients have an increased incidence of cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycaemia (HG), hyperinsulinaemia (HI) and combined HG+HI (induced using 24-hour infusion clamps) increases TF-PCA in healthy and type 2 DM (T2DM) subjects, but not in type 1 DM (T1DM) subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24-hour HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. In vivo, in healthy subjects, 24-hour HG + HI infusion increased TLR4 six-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hours). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects.

Protein disulphide-isomerase A2 regulated intracellular tissue factor mobilisation in migrating human vascular smooth muscle cells.

Protein-disulphide isomerase family (PDI) are an ER-stress protein that controls TF-procoagulant activity but its role in HVSMC migration and coronary artery disease remains to be elucidated. We aimed to investigate whether in human coronary smooth muscle cells (HVSMC) the ER-stress protein-disulphide isomerase family A member 2 (PDIA2) regulates tissue factor (TF) polarisation during migration and atherosclerotic remodeling. PDIA2 and TF were analysed by confocal microscopy, silenced by small interfering RNAs (siRNA) and their function analysed by transwell and migration assays in vitro and in vivo. PDIA2and TF co-localise in the front edge of motile HVSMC. Silencing PDIA2, as well as silencing TF, reduces migration. PDIA2 silenced cells show increased TF-rich microparticle shedding. In vivo cell-loaded plug implants in nude mice of PDIA2 silenced HVSMC together with microvascular endothelial cells showed a significant impairment in mature microvessel formation. PDIA2 and TF are found in remodelled atherosclerotic plaques but not in healthy coronaries. In conclusion, we demonstrate that TF is chaperoned by PDIA2 to the HVSMC membrane and to the cell migratory front. Absence of PDIA2 impairs TF intracellular trafficking to its membrane docking favoring its uncontrolled release in microparticles. TF-regulated HVSMC migration and microvessel formation is under the control of the ER-protein PDIA2.

Δ(9)-Tetrahydrocannabinol (THC) enhances lipopolysaccharide-stimulated tissue factor in human monocytes and monocyte-derived microvesicles.

BackgroundImmunomodulatory effects in humans of Δ9−Tetrahydrocannabinol (THC), the psychoactive component of marijuana are controversial. Tissue factor (TF), the activator of the extrinsic coagulation cascade, is increased on circulating activated monocytes and is expressed on microvesicles released from activated monocytes during inflammatory conditions, which perpetuate coagulopathies in a number of diseases. In view of the increased medicinal use of marijuana, effects of THC on human monocytes and monocyte-derived microvesicles activated by lipopolysaccharide (LPS) were investigated.FindingsPeak levels of TF procoagulant activity developed in monocytes or microvesicles 6 h following LPS treatment and were unaltered by THC. After 24 h of LPS stimulation, TF activity declined in control-treated or untreated cells and microvesicles, but persisted with THC treatment. Peak TF protein occurred within 6 h of LPS treatment independent of THC; by 24 h, TF protein declined to almost undetectable levels without THC, but was about 4-fold greater with THC. Steady-state TF mRNA levels were similar up to 2 h in the presence of LPS with or without THC, while 10-fold greater TF mRNA levels persisted over 3–24 h with THC treatment. Activation of MAPK or NF-κB pathways was unaltered by THC treatment and inflammatory cytokine IL-6 levels were unchanged. In contrast, TNF and IL-8 levels were enhanced by 20–50 %.ConclusionsTHC enhances TF expression in activated monocytes resulting in elevated procoagulant activity. Marijuana use could potentiate coagulopathies in individuals with chronic immune activation such as HIV-1 infection or inflammatory bowel disease.

Abstract 43: Recycling Controls Procoagulant and Cell Signaling Functions of Tissue Factor

Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell danger signals in form of high level of extracellular ATP trigger the macrophage P2X7 receptor leading to secretion of pro-inflammatory IL-1β and the release of pro-thrombotic tissue factor (TF) positive microparticles (MP). P2X7 receptor signaling uncouples the thioredoxin-thioredoxin reductase system and induces inflammasome activation leading to decryption of cell-surface TF and its trafficking onto filopodia for incorporation into MP. These MP have a distinct composition of thiol-regulated proteins and a high surface density of procoagulant PS. Besides its well-established function in IL-1β processing and secretion, we found an entirely unexpected role for caspase 1 in the release of TF from the final severing of highly thrombotic TF-positive MP. Thus, caspase 1 directly couples the release of pro-inflammatory and pro-thrombotic mediators. P2X7 activation not only causes MP release, but also activates cell surface TF that remains non-coagulant without secondary signals on primed macrophages. We observed that cell detachment rapidly induced full TF procoagulant activity, pointing to an important role of cell adhesion in the maintenance of a non-thrombotic state. Stable cell adhesion is controlled by integrin recycling pathways and the small GTPase arf6 is one of the key regulators of these processes. We found that blocking receptor internalization retains TF on the cell surface and markedly enhanced TF procoagulant activity. Under these conditions, TF was associated with integrin β1 and arf6, and importantly, P2X7 receptor activation promoted arf6-GTP hydrolysis to arf6-GDP, indicating interruption of this recycling pathway following stimulation. In addition, increasing arf6-GDP levels, either by pharmacological inhibitor or overexpression of an arf6-mutant, prevented TF-dependent signaling though protease activated receptor 2 (PAR2) and simultaneously increased cellular procoagulant TF activity in epithelial and in melanoma cells. These findings demonstrate an unexpected role for arf6-controlled recycling in the regulation of TF procoagulant and signaling functions.

IL‐13 Augments Compression‐Induced Exosomal Tissue Factor Release from Bronchial Epithelial Cells

Tissue factor (TF) is involved in multiple processes implicated in asthma, including coagulation, inflammation and angiogenesis. TF is released from well-differentiated human bronchial epithelial (HBE) cells in response to mechanical compression mimicking asthmatic bronchospasm. Here we investigated the combined effect of mechanical compression and exposure to the Th2 cytokine, IL-13, on the expression and release of TF from airway epithelial cells. Primary HBE cells were grown in air-liquid interface (ALI) culture for 14-17 days and were treated with IL-13 or vehicle and exposed to a trans-cellular pressure of 30 cmH20 for 3 hours. The pro-coagulant activity of basolateral media from HBE cells was significantly greater in compressed versus non-compressed cells, and was TF-dependent. Pretreatment of cells with IL-13 augmented compression-induced TF mRNA expression. Exposure to IL-13 alone did not affect the release of TF, but pretreatment with IL-13 augmented the compression-induced release of TF. Released TF was exclusively contained in the exosome fraction isolated by ultracentrifugation. Lastly, the TF level and pro-coagulant activity were significantly greater in BAL from mice challenged with ovalbumin versus PBS. Our data are consistent with the hypothesis that bronchoconstriction contributes to the generation of a lung microenvironment favoring asthma development. Compressive stress-mediated production and release of active TF from airway epithelial cells was augmented in the presence of the Th2 cytokine, IL-13.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium

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 IκBα 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/GSK3β 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.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.

Role of Tissue Factor in Mycobacterium Tuberculosis-induced Inflammation and Disease Pathogenesis

Tuberculosis, a chronic lung infection caused by Mycobacterium tuberculosis (M.tb), affects nearly one third of the world’s population. Clinical manifestations of TB include hypercoagulable states and thrombotic complications particularly disseminated intravascular coagulation and deep vein thrombosis. Tissue factor (TF) plays an important role in the initiation of inflammation-induced coagulation. Various bacterial infections induce aberrant expression of TF on vascular cells, which contributes to intravascular coagulation and exacerbation of inflammation. Studies have shown that either a genetic deficiency of TF or blockade of TF functional activity reduces coagulopathy, proinflammatory cytokine release and infection-associated mortality. In contrast, TF-dependent coagulation activation and fibrin deposition may be protective in host-defense against certain bacterial infections via reducing pathogen burden and limiting their dissemination. In vitro M.tb infection markedly upregulates TF expression and increases procoagulant activity of macrophages. However, it is not yet known whether TF expression has any functional significance in TB pathogenesis. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth and containment of infection. Wild-type C57BL6 (WT) and transgenic mice that express either very low levels of human TF (low TF, ~1% of WT) or high levels of human TF (HTF, ~100% of WT) in place of murine TF were infected with aerosol exposure of M.tb H37Rv. Mice were sacrificed 2 and 8 weeks post-infection. An evaluation of in vivo TF expression, coagulation activation, proinflammatory cytokines and tissue bacterial burden was performed. M.tb infection did not significantly alter overall TF expression and procoagulant activity in lungs of WT and HTF mice. Although not statistically significant, M.tb infection increased TF activity substantially in the lung homogenates in low TF mice. Nonetheless, TF expression levels in lungs of low TF mice, both uninfected and M.tb.-infected, was negligible as compared to WT and HTF mice. M.tb infection markedly increased TF expression in localized areas within the granulomas of WT and HTF mice. Interestingly, these intensely stained TF positive patches were also present in the granulomas of low TF mice after M.tb infection. The increased localized expression of TF in low TF mice may be responsible for the increased TF activity in the lung homogenates in low TF mice. M.tb infection was not accompanied by systemic and pulmonary activation of coagulation in WT and HTF mice. There was no change in the plasma thrombin-anti-thrombin complexes (TATc) upon M.tb infection in all three genotypes. Although, the bronchoalveolar lavage (BAL) TATc significantly increased (10-fold) after M.tb infection in the low TF mice, still the level was 15-50 folds lower than those in HTF and WT mice. The levels of TNF-α, IFN-γ, IL-6 and IL1-β increased upon M.tb infection but no significant differences in the cytokine profiles of BAL and total lung homogenates were observed among the genotypes. Higher expression of TF in the granuloma of WT and HTF correlated with the presence of small discrete regions of fibrin islands especially extending toward outer margin of the granuloma whereas little fibrin staining was seen in the granuloma of low TF mice. Despite, marked differences in fibrin generation in the granuloma, there were no significant differences in either lung bacterial burden or dissemination to liver and spleen. In summary, our data suggest that TF-mediated coagulation and/or signaling does not appear to contribute to host defense during experimental tuberculosis. However, it is difficult to completely eliminate a role for TF in M.tb. pathogenesis since M.tb. induced significant amount of TF expression in localized areas in the granuloma even in low TF mice. It is possible that this small amount of TF expressed within the granuloma may be sufficient to mediate local coagulant and signaling functions to facilitate M.tb. growth and dissemination. DisclosuresNo relevant conflicts of interest to declare.

BMP-7 induces TF expression in human monocytes by increasing F3 transcriptional activity

BackgroundBone morphogenetic protein (BMP)-7, a major regulator of bone metabolism, inhibits ectopic calcification in atherosclerotic plaques. We have recently reported that BMP-7 is also a potent inducer of tissue factor (TF) in human mononuclear cells (MNCs). While nuclear factor kappa beta (NF-kB) and activation protein-1 (AP-1) are the transcription factors essential for inducible expression of human TF gene (F3), the mechanisms responsible for TF induction by BMP-7 are not known. ObjectiveTo elucidate the molecular mechanisms governing BMP-7-triggered TF expression in human MNCs. MethodsHuman blood monocytes were stimulated with BMP-7 and western blotting, qRT-PCR, and flow cytometry studies were carried out to assess F3 expression; promoter studies were also performed using a panel of reporter constructs. Procoagulant TF activity was measured using a validated FXa generation assay. The significance of NF-kB transcriptional activity was verified via pharmacological inhibition. ResultsBMP-7 increased TF protein levels, procoagulant activity, surface presentation, and TF mRNA expression. This increase was accompanied by activation of NF-kB as evidenced by reduced IkB-α levels and elevated transcriptional activity of an NF-kB-sensitive reporter in transfected MNCs. Although treatment with BMP-7 also led to a strong phosphorylation of c-Jun, activation of AP-1 alone was not sufficient to induce TF expression: JSH-23, a potent and specific NF-kB inhibitor, completely blocked BMP-7-induced TF expression. ConclusionsWe report that BMP-7-dependent activation of TF in human MNCs is mediated via increased activity of NF-kB, leading to enhanced F3 transcription in human MNCs.

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.