Atherothrombosis: role of tissue factor; link between diabetes, obesity and inflammation

Atherothrombotic vascular disease is a complex disorder in which inflammation and coagulation play a pivotal role. Rupture of high-risk, vulnerable plaques with the subsequent tissue factor (TF) exposure is responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Tissue factor (TF), the key initiator of coagulation is an important modulator of inflammation. TF is widely expressed in atherosclerotic plaques and found in macrophages, smooth muscle cells, extracellular matrix and acellular lipid-rich core. TF expression can be induced by various stimulants such as C-reactive protein, oxLDL, hyperglycemia and adipocytokines. The blood-born TF encrypted on the circulating microparticles derived from vascular cells is a marker of vascular injury and a source of procoagulant activity. Another form of TF, called alternatively spliced has been recently identified in human and murine. It is soluble, circulates in plasma and initiates coagulation and thrombus propagation. Evidence indicates that elevated levels of blood-borne or circulating TF has been associated with metabolic syndrome, type 2 diabetes and cardiovascular risk factors and is a candidate biomarker for future cardiovascular events. Therapeutic strategies have been developed to specifically interfere with TF activity in the treatment of cardiovascular disease.     

Activation of blood coagulation in cancer: implications for tumour progression

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies.     

Regulation of angiogenesis by tissue factor cytoplasmic domain signaling

Hemostasis initiates angiogenesis-dependent wound healing, and thrombosis is frequently associated with advanced cancer. Although activation of coagulation generates potent regulators of angiogenesis, little is known about how this pathway supports angiogenesis in vivo. Here we show that the tissue factor (TF)-VIIa protease complex, independent of triggering coagulation, can promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. In this context, the TF cytoplasmic domain negatively regulates PAR-2 signaling. Mice from which the TF cytoplasmic domain has been deleted (TF Delta CT mice) show enhanced PAR-2-dependent angiogenesis, in synergy with platelet-derived growth factor BB (PDGF-BB). Ocular tissue from diabetic patients shows PAR-2 colocalization with phosphorylated TF specifically on neovasculature, suggesting that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR-2 signaling in angiogenesis. Targeting the TF-VIIa signaling pathway may thus enhance the efficacy of angiostatic treatments for cancer and neovascular eye diseases.     

组织因子与代谢性综合征的研究进展

组织因子(Tissue factor,TF)又称凝血因子III,是凝血因子VII/VIIa细胞表面的受体和辅因子,为外源性凝血的启动因子,在生理性止血、病理性血栓形成等发挥重要作用。近年临床研究发现,TF在代谢性综合征(Metabolic syndrome,MS)患者体内活性水平明显升高,并且与胰岛素抵抗、2型糖尿病、中心性肥胖、高血压、血脂紊乱等MS多种临床症候群密切相关。本文就近年国外相关进展作一综述,以期为针对TF这一作用靶点的MS病情演变预测及药物开发提供参考依据。     

Plexiform-like lesions and increased tissue factor expression in a rat model of severe pulmonary arterial hypertension

Severe pulmonary arterial hypertension (PAH) occurs in idiopathic form and in association with diverse diseases. The pathological hallmarks are distal smooth muscle hypertrophy, obliteration of small pulmonary arteriole lumens, and disorganized cellular proliferation in plexiform lesions. In situ thrombosis is also observed. A detailed understanding of the disease progression has been hampered by the absence of an animal model bearing all the pathological features of human disease. To create a model with these characteristics, we gave young (200-g) rats monocrotaline 1 wk following left pneumonectomy; controls with vehicle treatment or sham operation were also studied. In experimental rats, pulmonary arteries had distal smooth muscle hypertrophy and proliferative perivascular lesions. The lesions had a plexiform appearance, occurred early in disease development, and were composed of cells expressing endothelial antigens. Three-dimensional microangiography revealed severe vascular pruning and disorganized vascular networks. We found that expression of tissue factor (TF), the membrane glycoprotein that initiates coagulation, facilitates angiogenesis, and mediates arterial injury in the systemic circulation, was increased in the pulmonary arterioles and plexiform-like lesions of the rats. TF was also heavily expressed in the vessels and plexiform lesions of humans with pulmonary arterial hypertension. We conclude that plexiform-like lesions can be reproduced in rats, and this model will facilitate experiments to address controversies about the role of these lesions in PAH. Increased TF expression may contribute to the prothrombotic diathesis and vascular cell proliferation typical of human disease.     

Autophagy Mediates the Delivery of Thrombogenic Tissue Factor to Neutrophil Extracellular Traps in Human Sepsis

Background

Sepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation.

Methodology/Principal Findings

In this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling.

Conclusions/Significance

This study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis.     

Progesterone utilizes distinct membrane pools of tissue factor to increase coagulation and invasion and these effects are inhibited by TFPI

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.     

Initiation of coagulation by tissue factor

Tissue factor (TF) is an integral membrane glycoprotein which functions as an initiator of coagulation. Furthermore, it is probably the principal biological initiator of this essential hemostatic process. This article reviews the studies which form the basis for these assertions. The work on TF is traced from the 19th century discovery of the thromboplastic activity of tissues to the recent purification of the protein from bovine and human tissues and the isolation cDNA clones coding from human TF. The features of TF structure and function which tailor it to the role of initiator of the coagulation cascade are considered. For example, cell-surface TF and factor VII, the plasma serine proteases zymogen, form a proteolytic complex without prior proteolysis of either component. In addition, a kinetic model for the molecular mechanism of TF-initiated clotting is reviewed. The factors which control the expression of TF procoagulant activity by cultured cells are examined in light of the hypothesized role of TF in normal hemostasis. Also, the potential pathological consequences of aberrant TF expression, i.e., thrombosis and hemorrhage, are explored.     

Thrombophilia in mice expressing a tissue factor variant lacking its transmembrane and cytosolic domain

Mice with a targeted truncation in the gene encoding tissue factor of blood coagulation (TF) to eliminate the cytosolic domain and carrying a neo(R) cassette in intron 5 unexpectedly displayed severe spontaneous thrombosis in various vascular beds. Thrombosis was observed in heterozygous TF(+/neo) mice, causing death of over 50% of adults within 36 weeks of birth, and fulminantly exacerbating in pregnant females. Homozygous TF(neo/neo) mice were more severely affected and died within 7 weeks after birth. These TF(neo) mice primarily synthesized a mutant mRNA aberrantly spliced from exon 5 to neo(R), encoding an apparently non-vesicle-binding soluble TF lacking both the transmembrane and cytosolic domain, but still capable of blood coagulation induction. This severe thrombotic phenotype associated with the presence of a non-anchored soluble TF variant underscores the recently recognized significance of circulating TF for thrombus formation and development.     

Intracoronary beta-irradiation enhances balloon-injury-induced tissue factor expression in the porcine injury model

Intracoronary brachytherapy (ICBT) effectively reduces restenosis but is associated with late thrombosis. Since tissue factor (TF) is an important mediator of arterial thrombosis, we tested the hypothesis that ICBT results in persistently augmented TF expression. Coronary arteries from 12 pigs were randomized to: control (C; no injury), oversized balloon injury (BI), or BI followed by ICBT. Animals were sacrificed at 1, 7, 14, or 60 days postprocedure, and coronary arteries collected for expression analyses and immunostaining. ICBT-treated arteries had higher TF antigen and activity at all time-points compared to BI arteries (Western blot: 16 571 +/- 2090 vs 10 135 +/- 2939 densitometric units, p = 0.001; ELISA: 0.42 +/- 0.13 nM vs 0.25 +/- 0.14 nM, p = 0.001; TF activity assay: 0.303 +/- 0.11 nM vs 0.18 +/- 0.07 nM, p = 0.01; immunohistochemical staining: 30.6 +/- 6.6% vs 11.5% +/- 3.2%, p = 0.01). TF expression increased following BI, increased further following ICBT, and persisted for the duration of the study. We conclude that TF expression increases after BI, but is further increased and persists for a longer duration following ICBT, suggesting that a TF-mediated mechanism may play a role in late thrombosis following ICBT.     

Tissue factor upregulation drives a thrombosis-inflammation circuit in relation to cardiovascular complications

The extrinsic coagulation is recognized as an 'inducible' signalling cascade resulting from tissue factor (TF) upregulation by exposure to clotting zymogen FVII upon inflammation or tissue injury. Following the substantial initiation, an array of proteolytic activation generates mediating signals (active serine proteases: FVIIa, FXa and FIIa) that lead to hypercoagulation with fibrin overproduction manifesting thrombosis. In addition, TF upregulation plays a central role in driving a thrombosis-inflammation circuit. Coagulant mediators (FVIIa, FXa and FIIa) and endproduct (fibrin) are proinflammatory, eliciting tissue necrosis factor, interleukins, adhesion molecules and many other intracellular signals in different cell types. Such resulting inflammation could ensure 'fibrin' thrombosis via feedback upregulation of TF. Alternatively, the resulting inflammation triggers platelet/leukocyte/polymononuclear cell activation thus contributing to 'cellular' thrombosis. TF is very vulnerable to upregulation resulting in hypercoagulability and subsequent thrombosis and inflammation, either of which presents cardiovascular risks. The prevention and intervention of TF hypercoagulability are of importance in cardioprotection. Blockade of inflammation reception and its intracellular signalling prevents TF expression from upregulation. Natural (activated protein C, tissue factor pathway inhibitor, or antithrombin III) or pharmacological anticoagulants readily offset the extrinsic hypercoagulation mainly through FVIIa, FXa or FIIa inhibition. Therefore, anticoagulants turn off the thrombosis-inflammation circuit, offering not only antithrombotic but anti-inflammatory significance in the prevention of cardiovascular complications.     

Hypoxia primes endotoxin-induced tissue factor expression in human monocytes and endothelial cells by a PAF-dependent mechanism

Tissue factor (TF) is a glycoprotein which acts as a trigger of the coagulation cascade. TF expression may be induced at the surface of monocytes and endothelial cells by several stimuli including bacterial endotoxin (LPS) and cytokines (IL1β, TNFα) and there is a large body of evidence for the involvement of hypoxia as a primaring factor in the process leading to thrombosis. To define the molecular basis underlying this phenomenon, we evaluated the relative role of platelet activating factor (PAF). PAF primed human monocytes and human umbilical vein endothelial cells (HUVEC) for TF expression following exposure to E. coli LPS but was unable to enhance the induction of TF expression by IL1β. The priming effect of PAF with regard to LPS occurred in a time-and dose-dependent manner and was inhibited by the PAF receptor antagonist SR 27417. When HUVEC or monocytes were exposed to an hypoxic environment, a significant rise in LPS-induced TF expression was observed. Hypoxia had no effect on IL1-induced TF expression. The enhanced LPS-induced TF expression in both cell types was mediated by PAF as indicated by the inhibition obtained with SR 27417, added during hypoxia. Although the importance of hypoxia in the etiology of venous thrombosis has been acknowledged for a long time, evaluation of the relative importance of PAF in the process leading to thrombus formation is still lacking. Stasis-induced thrombosis performed in the rabbit jugular vein was enhanced in a dose-dependent manner by the prior i.v. administration of LPS (0.05 to 100 μg/kg, i.v.). SR 27417 administered simultaneously with LPS prevented thrombus formation with an ED50 value of 0.1 ± 0.04 mg/kg. These results therefore show that hypoxia promotes LPS-induced TF expression in HUVEC and human monocytes through a PAF-dependent mechanism in vitro and in vivo. © 1996 Wiley-Liss, Inc.     

Links between inflammation and thrombogenicity in atherosclerosis

Plaque disruption and subsequent thrombus formation play a critical role in the clinical manifestations of atherothrombosis. Vulnerable lesions are characterized by the existence of core rich in lipid, macrophages and tissue factor (TF). Plaque disruption facilitates the interaction between flowing blood with the inner components (TF) of disrupted atherosclerotic lesions triggering the coagulation cascade. TF, thrombin, platelets, fibrin and inflammatory cells are involved in this process of acute thrombus formation. This pathologic process is significantly accelerated by several "cardiovascular risk factors" such as diabetes, smoking, dyslipemia, etc. We will review on the role of TF, plaque cell apoptosis and blood thrombogenicity acting as a thread of inflammatory and prothrombotic mediators. We will also review the role of activated platelets as source for pro-inflammatory cytokines and enunciation of thrombotic process. Overall, we will try to emphasize the most recent understanding of the concepts involved in the interaction between inflammation and coagulation within the setting of atherothrombotic disease.     

Proteome of endothelial cell-derived procoagulant microparticles

Microparticles (MP) are small membrane vesicles that are released from cells upon activation or during apoptosis. Cellular MP in body fluids constitute a heterogeneous population, differing in cellular origin, numbers, size, antigenic composition and functional properties. MP support coagulation by exposure of tissue factor (TF), the initiator of coagulation in vivo. Moreover, MP may transfer bioactive molecules to other cells, thereby stimulating them to produce cytokines, cell-adhesion molecules, growth factors and TF, and modulate endothelial functions. However, a comprehensive characterization of the antigenic composition of MP has been poorly defined. This study describes the protein composition of endothelial cell (EC)-derived MP (EMP) using a proteomic approach. MS analysis indicated the presence of newly described protein such as metabolic enzymes, proteins involved in adhesion and fusion processes, members of protein folding event, cytoskeleton associated proteins and nucleosome. In conclusion, circulating EMP behave as an actual storage pool, able to disseminate blood-borne TF activity and other bioactive effectors, as confirmed by our experiments showing an increased procoagulant activity of EC exposed to EMP.     

N-methylnicotinamide inhibits arterial thrombosis in hypertensive rats.

There are few findings indicating that nicotinamide may potentially influence intravascular thrombosis. Interestingly, N-methylnicotinamide, one of the metabolites of nicotinamide - could be more potent than its parent compound. In the present study we have investigated the influence of N-methylnicotinamide on arterial thrombosis in normotensive and renovascular hypertensive rats. The contribution of platelets, coagulation and fibrinolytic systems in the mode of N-methylnicotinamide action was also determined. Furthermore, we examined the role of nitric oxide/prostacyclin in the mechanisms of N-methylnicotinamide action. N-methylnicotinamide, but not nicotinamide, administered intravenously into renovascular hypertensive rats developing electrically induced arterial thrombosis caused dose-dependent decrease of thrombus weight, collagen-induced platelet aggregation and plasma antigen/activity of plasminogen activator inhibitor - 1, without changing of occlusion time, routine coagulation parameters and plasma activity of tissue plasminogen activator. Indomethacin - an inhibitor of prostacyclin synthesis, completely abolished the antithrombotic and antiplatelet effect of N-methylnicotinamide, and the plasma level of 6-keto-PGF(1alpha) , prostacyclin metabolite, increased simultaneously with the inhibition of thrombus formation. Our study shows that N-methylnicotinamide via production/release of prostacyclin inhibits arterial thrombosis development. The antithrombotic effect of N-methylnicotinamide is accompanied by platelet inhibition and enhanced fibrinolysis, due to the decrease production of plasminogen activator inhibitor - 1.     

A simplified and high-throughput chromogenic assay for testing tissue factor-dependent procoagulant activity

Tissue factor (TF), the primary initiator of the coagulation cascade, plays a critical role in hemostasis and thrombosis, and inhibition of TF activity appears to be an attractive target for the treatment of cardiovascular diseases. However, few selective small-molecule inhibitors of TF are available, and the present assays for measuring TF activity are relatively expensive and complex. The authors present a simple and high-throughput chromogenic assay for screening TF inhibitors based on using commercial human prothrombin complex instead of purified coagulation factors, reducing the dosage, and performing with a one-stage procedure. In the optimized assay, <45 μL cell lysates was incubated with Tris-CaCl(2) buffer (pH 7.3) containing human prothrombin complex at 37°C for 15 min in 96-well or 384-well plates. Tris-EDTA buffer (pH 8.4) containing chromogenic substrate Xa was then added and the absorbance measured at 405 nm. This simplified assay was more sensitive or precise than some reported methods for TF procoagulant activities. Two known active compounds (curcumin and simvastatin) inhibiting TF activity were tested by the simplified assay to validate the screening method. Furthermore, berberine and cryptotanshinone suppressed TF activity induced by lipopolysaccharides in human monocytes by this assay and might be promising new TF inhibitors.     

Protease-activated receptor 2-dependent phosphorylation of the tissue factor cytoplasmic domain

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Pro-directed kinase target site Ser258 is dependent on prior phosphorylation of Ser253 by protein kinase C (PKC) alpha. TF phosphorylation is somewhat delayed and coincides with sustained PKCalpha activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKCalpha recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling.     

Angiotensin II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rats.

Although there are some in vitro evidence that angiotensin II (Ang II) may promote thrombosis, there is still no data concerning effect of Ang II on arterial thrombus formation. In the present study we have investigated the influence of Ang II on electrically induced arterial thrombosis in a common carotid artery of renovascular hypertensive rats. Furthermore, we examined if Ang II effect is mediated via AT1 receptor. We measured some coagulation and fibrinolytic parameters at the same time. Since platelets play crucial role in the initiation of arterial thrombosis their contribution in the mode of Ang II action was also determined. Intravenous infusion of Ang II caused significant increase in arterial thrombus weight, which was reversed by losartan, selective AT1 receptor antagonist. The prothrombotic effect of Ang II was accompanied by increase in haemostatic and decrease in fibrinolytic potential of rat plasma. While number of data has clearly demonstrated that Ang II can augment human platelets aggregation, at least in rats, platelets were not involved in the mechanism of Ang II action. Our study shows that Ang II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rat, therefore may be considered as a risk factor of myocardial infarction or stroke.     

A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways

Neutrophils and complement are key sentinels of innate immunity and mediators of acute inflammation. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways. To date, the potential cross-talk between innate immunity and thrombosis and the precise molecular pathway by which complement and neutrophils trigger the coagulation process have remained elusive. In this study, we demonstrate that antiphospholipid Ab-induced complement activation and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade. TF expression by neutrophils was associated with an enhanced procoagulant activity, as verified by a modified prothrombin time assay inhibited by anti-TF mAb. Inhibition studies using the complement inhibitor compstatin revealed that complement activation is triggered by antiphospholipid syndrome (APS) IgG and leads to the induction of a TF-dependent coagulant activity. Blockade studies using a selective C5a receptor antagonist and stimulation of neutrophils with recombinant human C5a demonstrated that C5a, and its receptor C5aR, mediate the expression of TF in neutrophils and thereby significantly enhance the procoagulant activity of neutrophils exposed to APS serum. These results identify a novel cross-talk between the complement and coagulation cascades that can potentially be exploited therapeutically in the treatment of APS and other complement-associated thrombotic diseases.