LPS-induced Tissue Factor Research Articles

PCSK9 aggravated carotid artery stenosis in ApoE-/- mice by promoting the expression of tissue factors in endothelial cells via the TLR4/NF-κB pathway

Atherosclerosis, a chronic inflammatory disease, is the most relevant cause of carotid artery stenosis. Vascular endothelial cells (ECs) play a significant role in the development of atherosclerosis. In this chronic inflammatory environment, we aimed to investigate whether PCSK9 could mitigate atherosclerosis progression by reducing tissue factor expression in ECs via in vivo and in vitro assays. In vivo, we investigated the effect of PCSK9 inhibition on preventing atherosclerotic lesion formation in ApoE-/- mice fed a western diet. The results showed that inhibiting PCSK9 could significantly downregulate the protein expression of tissue factor (TF) in ECs to reduce the area of atherosclerotic plaques. In vitro, we incubated human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS). We found that LPS-induced TF elevation was suppressed by a PCSK9 inhibitor at both the mRNA and protein levels and that the TLR4/NF-κB pathway was also suppressed by a PCSK9 inhibitor. With respect to plasma samples from patients with carotid artery stenosis, we also demonstrated that the expression of TF was positively correlated with that of PCSK9. Thus, in addition to regulating lipid metabolism, the regulation of endothelial cell TF expression through the TLR4/NF-κB pathway may be a potential mechanism of PCSK9 in promoting atherosclerotic carotid stenosis.

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.

Effects of Isoquercetin on Plasma Protein Disulfide Isomerase Activity and Extracellular Vesicle Tissue Factor Antigen/Activity in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) results from a single mutation in the β-globin gene that causes hemoglobin polymerization, red cell sickling, vascular injury, and recurrent vaso-occlusive crises (VOC). Vascular protein disulfide isomerase (PDI) is shown to be critical for thrombus formation by facilitating neutrophil recruitment, platelet activation and vascular fibrin deposition, all processes implicated in sickle vasculopathy. Since plasma PDI is elevated in SCD and cell-surface associated PDI could promote tissue factor (TF) dependent thrombin generation, we investigated the effects of Isoquercetin (IQ) in patients with steady state SCD. Methods: RBC surface associated PDI was quantified by western blotting. Inhibitory effect of IQ on LPS-induced TF expression on sickle monocytes in vitro was determined using flow cytometry. We also measured in vivo the effect of exposure of IQ or placebo for at least 28 days on plasma PDI protein, PDI reductase activity, total plasma extracellular vesicles (EVs), total plasma TF+ EVs, EV-TF-dependent procoagulant activity and markers of coagulation (D-dimer and Thrombin-antithrombin Complexes) using previously published methods. We used plasma from steady state SCD patients participating in a randomized controlled trial (NCT04514510) obtained at baseline and 28 days after exposure to either Isoquercetin 1000mg once daily (n=22) or placebo (n=22). The patients were in steady state, at least 60 days remote from VOC or blood transfusion and on a stable dose of hydroxyurea. The Wilcoxon rank-sum test was used to perform significance testing for differences in the percent change in outcomes between the Isoquercetin and placebo groups. Results: Sickle RBCs had higher cell-surface associated PDI compared to ethnic matched healthy controls (fold change 5.09; p=0.028). In confirmatory experiments with RBC ghosts, extracellular PDI was cell-surface associated. Pretreatment with Quercetin (the active metabolite of Isoquercetin) reduced LPS-induced cell-surface TF expression on sickle monocytes in vitro in a dose dependent manner (Fig 1). Following Isoquercetin administration, there was no change in the percent reduction in PDI inhibitory activity in the IQ group (mean: 6.6, 95%CI [-21.6, 34.9]) compared to placebo group (mean; -31, 95%CI [-94, 32.6], p=0.63). The percent change from baseline in PDI antigen also did not differ between the IQ or placebo groups (IQ mean: 1.2, 95%CI [-11.3, 13.6]; placebo mean: 12, 95%CI [-6, 30.4], p=0.52) (Fig 1). There was no correlation between the percent change in PDI activity and antigen from baseline after treatment with IQ (Spearman's Rho, IQ: r2 =0.28; p=0.20 placebo: r2=0.10; p=0.67). Unexpectedly, total plasma EVs showed no difference between the IQ (mean: 38.4; 95%CI [-4.6, 81.5]) and the placebo group (mean: 9, 95%CI [-18, 37.3]; p=0.27) (Table 1) suggesting that IQ has no obvious anti-inflammatory effects. Moreover, plasma EVs were predominantly derived from RBCs and platelets as published previously by us and others, indicating no membrane stabilizing effect on sickle RBCs and platelets. Inhibiting PDI reductase activity could allosterically reduce TF procoagulant activity, but this hypothesis was untestable because the plasma EV-TF dependent procoagulant activity in this patient population was undetectable, possibly reflecting effective disease control by hydroxyurea. Conclusions: The anti-inflammatory effects of flavonoid quercetin in vitro do not appear to translate into measurable clinical benefits in vivo following short term treatment with IQ in steady state SCD. Trials of higher dose IQ and/or prolonged exposure duration could yield greater inhibition of plasma PDI activity that might reveal detectable clinical antithrombotic effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bacitracin and Rutin Regulate Tissue Factor Production in Inflammatory Monocytes and Acute Myeloid Leukemia Blasts

Simple SummaryAberrant tissue factor (TF) expression by transformed myeloblasts and inflammatory monocytes contributes to coagulation activation in acute myeloid leukemia (AML). TF procoagulant activity (PCA) is regulated by protein disulfide isomerase (PDI), an oxidoreductase with chaperone activity, but its specific role in AML-associated TF biology is unclear. Here, we provide novel mechanistic insights into this interrelation. We show that bacitracin and rutin, two pan-inhibitors of the PDI family, prevent lipopolysaccharide (LPS)-induced monocyte TF production under inflammatory conditions and constitutive TF expression by THP1 cells and AML blasts, thus exerting promising anticoagulant activity. Downregulation of the TF protein was mainly restricted to its non-coagulant, cryptic pool and was at least partially regulated on the mRNA level in LPS-stimulated monocytes. Collectively, our study indicates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with the most abundant PDI being a promising therapeutic target in the management of AML-associated coagulopathies.Aberrant expression of tissue factor (TF) by transformed myeloblasts and inflammatory monocytes drives coagulation activation in acute myeloid leukemia (AML). Although regulation of TF procoagulant activity (PCA) involves thiol-disulfide exchange reactions, the specific role of protein disulfide isomerase (PDI) and other thiol isomerases in AML-associated TF biology is unclear. THP1 cells and peripheral blood mononuclear cells (PBMCs) from healthy controls or AML patients were analyzed for thiol isomerase-dependent TF production under various experimental conditions. Total cellular and membrane TF antigen, TF PCA and TF mRNA were analyzed by ELISA, flow cytometry, clotting or Xa generation assay and qPCR, respectively. PBMCs and THP1 cells showed significant insulin reductase activity, which was inhibited by bacitracin or rutin. Co-incubation with these thiol isomerase inhibitors prevented LPS-induced TF production by CD14-positive monocytes and constitutive TF expression by THP1 cells and AML blasts. Downregulation of the TF antigen was mainly restricted to the cryptic pool of TF, efficiently preventing phosphatidylserine-dependent TF activation by daunorubicin, and at least partially regulated on the mRNA level in LPS-stimulated monocytes. Our study thus delineates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with PDI being a promising therapeutic target in the management of AML-associated coagulopathies.

LPS-induced expression and release of monocyte tissue factor in patients with haemophilia

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.

Alterations in Sphingomyelin Metabolism Influences Tissue Factor Procoagulant Activity and the Release of TF-Positive Microvesicles

Tissue factor (TF), an integral membrane glycoprotein, is a cofactor for coagulation factor VIIa (FVIIa) and primary cellular initiator of the coagulation. Upon vascular injury or in disease conditions, blood comes in contact with TF, and the formation of TF-FVIIa complex initiates activation of the coagulation cascade. While TF is critical for the maintenance of hemostasis, aberrant expression of TF activity could lead to thrombotic disorders. Typically, most of TF on cell surfaces exist in a cryptic, coagulant inactive state, and an "activation" step (decryption) is essential for the transformation of cryptic TF to prothrombotic TF. Our recent studies showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells. The hydrolysis of SM, by either bacterial sphingomyelinase (bSMase) or acid-sphingomyelinase (ASMase) translocated from lysosomes to the outer leaflet in response to ATP, LPS or cytokine stimulation, increased TF activity on intact cells without altering TF protein levels. SM hydrolysis also led to the release of TF+ microvesicles (MVs). Inhibition of ASMase by functional inhibitors blocked LPS-induced TF procoagulant activity without impairing LPS-induced TF antigen levels in both in vitro and in vivo model systems. SM levels in the plasma membrane are regulated primarily by SM synthesizing enzymes, such as sphingomyelin synthases (SMS) 1 and 2 or SM hydrolyzing enzymes, such as ASMase and neutral SMases (nSMase). Many disease conditions, including diabetes, ischemia/hypoxia, and cancer, alter SM metabolism by altering the activities of the above enzymes. These diseases are also known to have increased thrombotic risk. To investigate the importance of SM metabolism in regulating TF procoagulant activity through TF encryption and decryption, we either overexpressed or silenced the enzymes involved in SM metabolism and determined their effect on TF procoagulant activity on intact cells and the release of TF+ MVs. Human monocyte-derived macrophages (MDMs) or human embilical vein endothelial cells (HUVEC) were chosen as cell model systems. In the first set of experiments, MDMs were transfected with adenovirus encoding SMS1, SMS2, or both to overexpress SMS. Analysis of SM levels in the outer leaflet by confocal microscopy and flow cytometry using SM specific binding protein (lysenin) revealed that overexpression of SMS1 or SMS2 increased SM levels in the outer leaflet. Measurement of TF activity on intact cells showed that overexpression of either SMS1 or SMS2 reduced both basal TF activity and the extent of increased TF activity following ATP or bSMase treatment. Overexpression of SMS1 or SMS2 also decreased the release of TF+ MVs. Overexpression of SMS1 or SMS2 had no significant effect on TF antigen levels. In the next set of experiments, MDMS were transfected with control scrambled RNA (scRNA) or siRNA specific for ASMase, nSMase1, nSMase2, or nSMase3. As expected from our earlier studies, ASMase silencing attenuated both basal and ATP-induced increased TF activity in MDMs. In case of nSMases, the knock-down of nSMase2 or nSMase3, but not nSMase1, reduced basal TF activity as well as ATP-induced TF decryption in MDMs. Analysis of SM levels in the outer leaflet showed that silencing of ASMase, nSMase2, or nSMase3 enhanced the SM content. The knock-down of either ASMase or nSMases did not affect TF antigen levels. In additional studies, HUVECs were transfected with control scRNA or siRNA specific for nSMase1, nSMAse2, or nSMase3. Forty eight hour post-transfection, HUVECs were stimulated with TNFα (10 ng/ml) plus IL-1β (10 ng/ml) for 6 h to induce TF expression. Analysis of cell surface TF activity showed that silencing nSMase2 or nSMase3, but not nSMase1, attenuated TNFα+IL-1β-induced TF procoagulant activity without decreasing TNFα+IL-1β-induced TF antigen levels. Overall, our data support the hypothesis that alterations in SM metabolism regulate TF procoagulant activity through encryption and decryption. Disclosures Rao: Takeda: Research Funding.

Anti-\u03b22-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies exert similar pro-thrombotic effects in peripheral blood monocytes and endothelial cells

PurposeThe introduction of the anti-phosphatidylserine/prothrombin (aPS/PT) antibodies among the routinely investigated anti-phospholipid (aPL) antibodies led to an improvement in anti-phospholipid syndrome (APS) laboratory diagnostic performance; however, their pathogenic mechanism is still substantially undefined. To support clinical data and future inclusion as possible new criteria antibodies, we designed a head-to-head study to directly compare the procoagulant effects sustained in vitro by aPS/PT to those sustained by anti-β2-glycoprotein I (aβ2GpI) domain 1-specific antibodies.MethodsBlood donors-derived monocytes and endothelial cells (HUVEC) were stimulated with lipopolysaccharides (LPS) alone or in combination with the IgG fractions isolated from the serum of six APS patients, positive only for aβ2GpI or for aPS/PT antibodies. As control, cells were incubated with LPS plus the IgG isolated from blood donors. Tissue factor (TF) mRNA expression was measured after four hours incubation by real-time PCR. Nitric oxide (NO) levels were measured in cells supernatant after 16 h incubation by colorimetric assay.ResultsaPS/PT and aβ2GpI IgG antibodies fractions showed comparable ability to enhance LPS-induced TF mRNA expression, either in monocytes and in HUVEC. Compared to LPS alone, we found that NO levels are strongly overproduced in HUVEC treated with LPS plus aβ2GpI and aPS/PT IgG fractions.ConclusionsOur data support the significant and independent role of aPS/PT in the pathogenesis of the thrombotic events in APS patients, possibly adding new light to the therapeutic management of cases characterized by the sole presence of aPS/PT IgG antibodies.

The effect of high glucose on the inhibitory action of C21, a selective AT2R agonist, of LPS-stimulated tissue factor expression in human mononuclear cells.

BackgroundIntimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively).ResultsHG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG.ConclusionsHG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathways.

Yes-associated protein (YAP) signaling regulates lipopolysaccharide-induced tissue factor expression in human endothelial cells

Sepsis-induced acute lung injury (ALI) is characterized by fibrin deposition, which indicates the local activation of coagulation. Tissue factor (TF), expressed in the pulmonary microvasculature, acts as a critical initiator of blood coagulation and ALI in sepsis. The molecular mechanism of lipopolysaccharide (LPS)-induced TF expression in endothelial cells (ECs), however, has not been determined. In this study, we implicate the Rho-associated protein kinase (ROCK)/Yes associated protein (YAP)/early growth response (Egr-1) signaling pathway in LPS-induced TF expression in vitro and in sepsis-induced ALI in vivo. Human umbilical vein ECs incubated with LPS were pretreated with or without the ROCK inhibitor Y-27632, a YAP small, interfering RNA (siRNA) and an Egr-1 siRNA. ROCK, YAP and Egr-1 signaling-induced protein expression was investigated by Western blot. The LPS-induced activation of YAP was analyzed by an immunofluorescent assay. Furthermore, we intratracheally injected YAP siRNA to assess septic ALI in mice by hematoxylin and eosin staining. LPS rapidly induced ROCK activation and increased TF expression in ECs. LPS caused YAP shuttling into the nuclei of ECs and combined with Egr-1 via the activation of ROCK. Furthermore, the LPS-mediated TF expression increase was prevented by ROCK inactivation, YAP knockdown and Egr-1 depletion, suggesting that LPS-induced TF expression is closely associated with the ROCK/YAP/Egr-1 signaling pathway in ECs. Finally, an intratracheal injection of YAP siRNA relieved lung injury in septic mice. This study not only suggests that ROCK/YAP/Egr-1 signaling regulates TF expression after stimulation with LPS in ECs, but it also indicates that LPS-induced activation of YAP signaling plays an important role in septic ALI in mice. Our findings provide a new insight into the pathogenic mechanism of TF expression, which is closely linked to septic ALI, and YAP signaling is considered to be a novel target for therapeutic intervention under septic conditions.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-β2-glycoprotein I (anti-β2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of β2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

Procoagulant and proinflammatory effects of red blood cells on lipopolysaccharide‐stimulated monocytes

We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Endothelial cells but not platelets are the major source of Toll-like receptor 4 in the arterial thrombosis and tissue factor expression in mice.

It is known that Toll-like receptor (TLR)-4 plays an important role in myocardial infarction and atherothrombosis. The role of TLR-4 in arterial thrombosis is undefined. Both TLR-4-deficient (TLR-4(-/-)) and wild-type (WT) mice were subjected to FeCl3 carotid artery injury, and the time required to form an occlusive thrombus was measured. The mean time to occlusion in TLR-4(-/-) mice was significantly greater than that in WT mice after injury (303 ± 32 vs. 165 ± 34 s, P < 0.05). Furthermore, when we used a WT or TLR-4(-/-)-derived platelet reinfusion in a platelet depletion/reinfusion procedure, there was no significant change in the occlusion time and tissue factor (TF) activity in injured arteries between WT mice and platelet-depleted WT mice. Similarly, no significant difference was observed between TLR-4(-/-) mice and platelet-depleted TLR-4(-/-) mice for the WT or TLR-4(-/-)-derived platelet reinfusion. However, TF expression and activity were significantly reduced in the vascular wall of TLR-4(-/-) mice compared with WT mice. In vivo, lipopolysaccharide accelerated the occlusion time in WT mice but not TLR-4(-/-) mice. In vitro, LPS-induced TF activity was reduced in endothelial cells of TLR-4(-/-) mice relative to WT mice. The data demonstrate that TLR-4 contributes to arterial thrombosis formation in vivo and causes increased TF expression and activity in vitro. The results further suggest that the stimulation is mainly derived by endothelial cells but is not due to platelet-derived TLR-4.

Compound 21, a selective angiotensin II type 2 receptor agonist, downregulates lipopolysaccharide-stimulated tissue factor expression in human peripheral blood mononuclear cells.

Intricate interrelationships connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally active angiotensin II, a proinflammatory agent with direct TF-stimulating properties mediated by the angiotensin II type 1 receptor (AT1R)s. However, angiotensin II also stimulates angiotensin II type 2 receptor (AT2R)s and they may as well contribute to TF expression, a possibility in need of further evaluation. We investigated the effect of C21, a highly specific AT2R agonist, on TF antigen (ELISA), procoagulant activity (PCA, one-stage clotting assay) and TF-mRNA (real-time PCR) in peripheral blood mononuclear cell (PBMC)s activated by lipopolysaccharide (LPS), a pro-inflammatory and procoagulant stimulus. C21 downregulated LPS-stimulated TF antigen, PCA and TF mRNA, an effect abolished by PD123 319, a selective AT2R antagonist, and left unchanged by omesartan, a selective AT1R antagonist. PD123 319 per se did not affect LPS-induced TF expression while omesartan inhibited and BAY 11-7082, a specific NFκB inhibitor, abolished endotoxin-activated procoagulant activity (PCA). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated PBMCs, a finding consistent with the existence in PBMCs of AT2Rs whose stimulation attenuates inflammation-mediated procoagulant responses. The data open insofar unexplored and potentially relevant facets to our understanding of the complex links connecting angiotensin II to inflammation and coagulation.

Inhibition of PI3K contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity (INC6P.340)

Abstract Nanoparticles are increasingly used in biomedical applications due to their ability to change biodistribution, increase safety and improve efficacy of drugs. However, application of engineered nanomaterials in medicine is often halted by safety concerns. Endotoxin contamination is very common in nanomaterials and there is an increasing number of studies reporting that certain nanoparticles can exaggerate endotoxin-mediated inflammation. We studied the relationship between physicochemical properties of polyamidoamine (PAMAM) dendrimers and their ability to exaggerate LPS-induced procoagulant activity (PCA) of human leukocytes in vitro. The PCA is phospholipid-protein complex composed of tissue factor (TF) and phosphotidylserine (PS). We found that cationic but not anionic or neutral PAMAM dendrimers, increase LPS-induced leukocyte PCA. Cationic PAMAM dendrimers enhanced both LPS-induced TF expression and PS exposure on the cell surface. The mechanism of LPS-induced PCA exaggeration by PAMAM dendrimers is not mediated by the electrostatic interaction between cationic dendrimers and anionic LPS, calcium influx and secondary messengers released by dendrimer activated platelets. We hypothesize that dendrimers exaggerate LPS-mediated PCA through the inhibition of negative regulation of inflammation. Specifically, we demonstrate that cationic PAMAM dendrimers inhibit phosphoinositol-3-kinase (PI3K), a negative regulator of TF expression in response to endotoxin.

Pro- and anti-inflammatory effects of histamine on tissue factor and TNFα expression in monocytes of human blood

Histamine is classified as an inflammatory mediator and has been reported to have anti- as well as pro-inflammatory properties. The aim of this study was to explore the role of histamine on the production of LPS-induced tissue factor (TF) activity and TNFα in monocytes of whole blood in the absence and presence of TNFα or PMA. Human blood anticoagulated with Fragmin was subjected to stimulation by LPS in the presence and absence of TNFα or PMA and various concentrations of histamine. Tissue factor (TF) activity was measured in lyzed cells after isolation of mononuclear cells whereas TNFα was quantified in plasma after centrifugation of cells. Histamine gave a dose dependent inhibitory effect on LPS-induced TF activity in monocytes of whole blood, with a 50% reduction at 0.033 μM. A similar effect was seen when the blood cells were stimulated with the combination of LPS and TNFα although TNFα enhanced LPS-induced TF activity almost two fold. In contrast, when blood was incubated with LPS and PMA in whole blood, histamine gave a significant rise in TF activity at 0.01 μM and 0.33 μM histamine. The effect of histamine was less at 0.1 μM or higher concentrations giving a biphasic profile. Contrary to the effect of histamine on LPS plus PMA induced TF activity, histamine caused a significant reduction in TNFα albeit less than in the absence of PMA. Intake of aspirin caused a significant rise in LPS-induced TF activity that was almost abolished by histamine at 0.033 μM. Our study shows that histamine has an anti-inflammatory effect on LPS and LPS/TNFα stimulated monocytes of whole blood. In contrast when blood cells are activated by a combination of LPS and PMA whereby PKC is activated, histamine has a procoagulant/pro-inflammatory effect through enhancement of TF activity expression.

Mechanical Stretch Inhibits Lipopolysaccharide-induced Keratinocyte-derived Chemokine and Tissue Factor Expression While Increasing Procoagulant Activity in Murine Lung Epithelial Cells

Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.

Mycobacterium tuberculosis Infection and Tissue Factor Expression in Macrophages

A number of earlier studies reported the occurrence of thrombotic complications, particularly disseminated intravascular coagulation and deep vein thrombosis, in tuberculosis (TB) patients. The aberrant expression of tissue factor (TF), the primary activator of coagulation cascade, is known to be responsible for thrombotic disorders in many diseases including bacterial infections. Further, expression of TF by cells of the monocyte/macrophage lineage is also shown to contribute to the development and progression of local and systemic inflammatory reactions. In the present study, we have investigated whether Mycobacterium tuberculosis (Mtb) infection induces TF expression in macrophages, and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. The data presented in the manuscript show that both live virulent Mtb and γ-Mtb treatments markedly increased TF activity in macrophages, predominantly in the CD14+ macrophages. Detailed studies using γ-Mtb showed that the increased TF activity in macrophages following Mtb treatment is the result of TF transcriptional activation. The signaling pathways of TF induction by Mtb appears to be distinct from that of LPS-induced TF expression. Mtb-mediated TF expression is dependent on cooperation of CD14/TLR2/TLR4 and probably yet another unknown receptor/cofactor. Mtb cell wall core components, mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) were identified as factors responsible for induction of TF in the order of mAGP>PIM6>LM. A direct contact between bacteria and macrophage and not Mtb-released soluble factors is critical for TF induction by Mtb. In summary, our data show that Mtb induces TF expression in macrophages and Mtb signaling pathways that elicit TF induction require cooperation of multiple receptors, co-receptors/co-factors including Toll-like receptors. The importance of TF in granuloma formation and containment of Mtb is discussed.

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

IntroductionHeme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects. Materials and MethodsHUVECs were pretreated with 50μM CORM-2 for 3hours, and stimulated with tumor necrosis factor-α (TNF-α, 10ng/ml) for an additional 0–5hours. PBMCs were pretreated with 50–100μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10ng/ml) for additional 0–9hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively. ResultsPretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs. ConclusionsCORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.

High glucose potentiates and renin-angiotensin blockade downregulates LPS-induced tissue factor expression in human mononuclear cells

BackgroundIntimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (ANG) II, the effector arm of the Renin Angiotensin System (RAS). The RAS, in turn, plays a pathophysiological role in diabetes, a proinflammatory state to which elevated glucose, the disease hallmark, contributes by activating key signalling pathways and increasing the cellular content of RAS components. AimsTo evaluate the effect of high glucose concentrations on TF antigen (Ag) expression and procoagulant activity (PCA) in lipopolysaccharide(LPS)-primed human mononuclear cell(MNC)s and to test whether pharmacological RAS blockade modifies that pattern. MethodsLPS-activated MNCs exposed to increasing D-glucose (from 5.5 to 50mM) in absence or presence of aliskiren, a renin inhibitor, zofenopril, an ANG converting enzyme inhibitor, and olmesartan, an ANGII type I receptor blocker. PCA was assessed by one-stage clotting assay and TF antigen expression by ELISA. ResultsIncreasing ambient glucose (range 5.5–50mM) potentiated LPS-induced PCA and TF Ag expression. Aliskiren, zofenopril and olmesartan downregulated those responses but the efficacy of the former decreased by ascending drug concentration while both zofenopril and olmesartan showed an opposite behaviour. TF Ag expression modulation by RAS blockade was stronger in 50 than 5mM ambient glucose. ConclusionsHigh glucose potentiates the procoagulant action of LPS in human MNCs and RAS blockers downregulate that response possibly as a reflection of the underlying involvement of the system in that mechanism.

Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1–1000ng/mL) for various times. Recombinant IL-10 (10–5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.