Tissue factor pathway inhibitor on circulating microparticles in acute myocardial infarction

In acute myocardial infarction (AMI), increased Tissue Factor (TF) expression on circulating monocytes and microparticles (MP) may contribute to thrombotic events. Because surfacebound Tissue Factor Pathway Inhibitor-1 (TFPI) inhibits TF activity on monocytes and endothelial cells decreased TFPI expression may reinforce the procoagulant activity of circulating MP. Aim of the study was to analyze TFPI expression and TF activity after stenting and thrombolysis inAMI. Thirty-nine patients of a randomized study comparing intravenous thrombolysis (n=19) and stenting (n=20) were included. Before and after therapy blood samples for analysis of MPs, TF antigen and activity, prothrombin fragment F1+2 and D-dimer were obtained. TFPI expression on TF positive MPs was decreased after thrombolysis but not after stenting. In contrast, TF plasma levels and TF positive MP remained unchanged in both treatment groups. After thrombolysis increased D-dimer and F1+2 plasma concentrations indicated activation of fibrinolysis and coagulation. Significance of MPTFPI for inhibition of TF activity was measured using inhibitory TFPI antibodies. Membrane-associated TFPI inhibited TF activity on circulating MPs. After thrombolysis inhibition of TF activity by TFPI was decreased as compared to stenting. Correlation of circulating TF with F1+2 only after thrombolysis, suggests a role for TF-induced activation of coagulation after thrombolysis. Enhanced TF activity on circulating MPs in AMI is inhibited by endogenous surface-boundTFPI. After thrombolysis but not after stenting MPTFPI is degraded and may induce thrombin generation due to unopposed tissue factor activity. Anti-TF therapies during thrombolysis may reduce thrombin generation in AMI.     

The expression of tissue factor and tissue factor pathway inhibitor in aortic smooth muscle cells is up-regulated in synthetic compared to contractile phenotype

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes. Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs. A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.     

Apoptosis as a determinant of atherothrombosis

Clinical manifestations of atherosclerosis are the consequences of atherosclerotic plaque rupture that triggers thrombus formation. Tissue factor (TF) is a key element in the initiation of the coagulation cascade and is crucial in thrombus formation following plaque disruption. TF activity is highly dependent on the presence of phosphatidylserine (PS), an anionic phospholipid that is redistributed on the cell surface during apoptotic death conferring a potent procoagulant activity to the apoptotic cell. Apoptosis occurs in the human atherosclerotic plaque and shed membrane apoptotic microparticles rich in PS are produced in considerable amounts within the lipid core. These microparticles carry almost all TF activity and are responsible for the procoagulant activity of the plaque. Moreover, luminal endothelial cell apoptosis might be responsible for thrombus formation on eroded plaques without rupture. Apoptosis might also play a major role in blood thrombogenicity via circulating procoagulant microparticles that are found at high levels in patients with acute coronary syndromes.     

Microparticles from apoptotic vascular smooth muscle cells induce endothelial dysfunction, a phenomenon prevented by beta3-integrin antagonists

Fragile atherosclerotic plaques are rich in apoptotic smooth muscle cells (SMCs) and macrophages, generating microparticules (MPs) which accumulate locally and may be released in blood in case of mechanical or spontaneous plaque disruption. Besides being highly procoagulant, this material may interact with downstream endothelium. Using a model of mouse aorta vaso-reactivity, we have investigated the effects of apoptotic MPs prepared in vitro from Fas-ligand sensitive SMCs. Short-term preincubation of aorta rings with the MPs dose-dependently reduced the vasodilatory response to acetylcholine dependent on the endothelium. This effect was prevented by the addition of abxicimab or eptifibatide, indicating a role for a beta3 integrin in this process. We further investigated its mechanism using cultured endothelial cells. The MPs were found to bind to the cells and to inhibit the production and the release of nitric oxide (NO) in response to bradykinin. This phenomenom was redox sensitive, independent of the generation of activated coagulation proteases, and was abrogated when the MPs were pretreated by trypsin. The metabolic effects of MPs were prevented by the addition of eptifibatide. Taken together, these results suggest a potential, platelet-independent, mechanism for the improvement of microvascular perfusion observed with beta3-integrin antagonists.     

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology

Background

Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized.

Objective

To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques.

Methods

We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n = 20) or echogenic (n = 20) carotid plaques, or controls without carotid atherosclerosis (n = 20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression.

Results

TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p = 0.007), and positive association between serum apoB and monocyte surface TFPI expression (p = 0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio.

Conclusions

Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.     

Molecular cloning, characterization and expression of cDNA for rabbit brain tissue factor.

Tissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factor VII or VIIa. TF has been identified in atherosclerotic plaques and may possibly trigger thrombosis after spontaneous plaque rupture as seen in acute myocardial infarction or angioplasty. We have previously developed an atherosclerotic rabbit model for study of the acute and chronic outcomes following angioplasty. As a first step in developing inhibitors of TF, we have isolated and characterized a rabbit cDNA coding for the mature TF. The sequence comparison of rabbit TF cDNA with those of human and mouse TFs show considerable similarity at both the nucleotide and amino acid levels. The TF cDNA when expressed in E. coli demonstrates a procoagulant activity comparable to that of native rabbit brain TF. The TF activity can be blocked by a polyclonal antibody against rabbit TF.     

Atorvastatin and thrombogenicity of the carotid atherosclerotic plaque: the ATROCAP study

Statins appear to have beneficial effects on fibrous cap stabilisation but their effects on plaque thrombogenicity have not been reported. To evaluate the thrombogenicity of human carotid plaques before and after atorvastatin treatment, 59 patients with bilateral carotid stenosis eligible for two-step carotid endoarterectomy (CEA) were randomly assigned to atorvastatin, 20 mg/day, or placebo. Histological and immunohistochemical analyses, Tissue Factor (TF), Tissue Factor Pathway Inhibitor (TFPI) antigens (Ag) and TF activity were determined in endoarterectomy specimens obtained at baseline and after treatment. Mean TFAg and TFPIAg levels from plaques removed at the first CEA were 55 +/- 56 and 32 +/- 26 pg/mg. After placebo, TFAg and TFPIAg content was higher in the second than the first CEA. Plaques removed at the second CEA from atorvastatin-treated patients had a lower macrophage content than plaques at the first CEA. TFAg and TFPIAg levels, and TF activity in plaques after atorvastatin treatment were lower (respectively 29, 18% and 56%) than after placebo. These findings indicate that atorvastatin reduce the inflammatory/thrombotic phenotype of carotid plaque, suggesting that these drugs may indeed have a beneficial effect on cerebrovascular events.     

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.     

Tissue factor/TFPI and blood cells

Vascular injury-induced access of blood to tissue factor (TF) leads to the formation of a TF-FVII/FVIIa complex and the triggering of blood coagulation. The activated TF-dependent pathway is regulated by Tissue Factor Pathway Inhibitor (TFPI), which binds and inhibits FXa, but more importantly forms an inactive quaternary complex with TF-FVIIa-FXa, effectively shutting off the TF activity. The old view of TF residing in extravascular sites exclusively has recently been challenged by several reports on TF expression in various blood cells. The latter arena has unfortunately been marred by many contradictions, apparently related to inferior tools and/or study design, notably the widespread use of antibodies with inferior and misleading specificity and TF activity assays of low sensitivity/specificity. Our own studies along with many other reports, compels the conclusion that in blood of healthy individuals TF is exclusively associated with and expressed in circulating monocytes. In this short review the distribution of TF and TFPI in blood is discussed.     

Postprandial elevation of tissue factor antigen in the blood of healthy adults

Atherosclerosis is a dynamic disease involving lipid metabolism, inflammation and thrombosis. A key factor in thrombosis is tissue factor, a small transmembrane glycoprotein. Tissue factor binds FactorVIIa, and this complex converts Factor X to Factor Xa, leading to thrombin generation and fibrin formation. Inhibition of this pathway is by tissue factor pathway inhibitor (TFPI). Tissue factor is found sequestered within atherosclerotic plaques, and plaque rupture allows tissue factor exposure to the circulation, leading to formation of a thrombus. Tissue factor is also associated with membrane microparticles in the circulation, most likely released from monocytes activated by an inflammatory event. We hypothesize that consumption of a typical western diet that is moderate in fat content leads to elevated levels of circulating tissue factor that may act as a marker of a prothrombotic state. Healthy volunteers, aged 18-55, consumed a moderate (40%) fat meal, with blood taken before and 3.5 and 6 h after the meal. Plasma was isolated and assayed for plasma triglycerides, tissue factor, thrombin antithrombin (TAT) complexes, TFPI and TNFalpha. The levels of circulating tissue factor increased 56% (from 78 pg/ml to 120 pg/ml) 3.5 h after the meal. Levels decreased, but had not returned to baseline 6 h postprandially. No significant differences in TAT, TFPI and TNFa levels were observed postprandially. These results demonstrate increased tissue factor levels in individuals who consumed a moderate fat diet. This suggests that the typical western diet may play a larger role in cardiovascular disease than merely altering lipid profiles.     

Serum lipids and regulation of tissue factor-induced coagulation in middle-aged men

Formation of an occlusive thrombus by exposure of tissue factor (TF) to circulating blood and subsequent triggering of coagulation by TF-activated factor VII (FVIIa) complexes on ruptured atherosclerotic plaques is thought to be a key event in myocardial infarction. Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of TF-induced coagulation in which the anticoagulant function most probably is restricted to free TFPI in human plasma. The present study was undertaken to assess the interrelations between serum lipids and components of TF-induced coagulation in 234 apparently healthy men aged 36-56 years recruited from the general population. Plasma free TFPI antigen (Ag) was positively correlated (P < .001) with total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, apolipoprotein B (apoB-100), fibrinogen, total amount of FVII (FVIIam), coagulation activity of factor VII (FVIIc), and FVIIa. The significant predictors for free TFPI Ag were total cholesterol, triglycerides, fibrinogen, FVIIc, and age, which explained 33% of the plasma variation in free TFPI Ag assessed by multiple regression analysis. A highly significant (P < .0001) linear trend for increase in atherogenic lipids (i.e., total cholesterol and triglycerides), FVII (i.e., FVIIc and FVIIa), and fibrinogen across quartiles of TFPI Ag was demonstrated after adjustment for confounders. These findings may indicate a compensatory increase in plasma free TFPI with lipid and hemostatic risk factors for atherothrombotic diseases in healthy middle-aged men.     

Effects of heparin and related sulfated polysaccharides on tissue factor expression induced by mitogenic and non-mitogenic factors in human vascular smooth muscle cells.

Smooth muscle cells (SMCs) of the intima are generally quiescent and non proliferative. Their proliferation due to different stimulations occurs in myointimal hyperplasia and is regularly present in atherogenesis or after transluminal angioplasty leading to vascular occlusive stenosis. In the course of these pathologies, the Tissue Factor (TF) synthesis was upregulated and rapidly expressed at the membrane of the SMCs. Heparin is known to inhibit SMCs proliferation induced by FCS. We evaluated the inhibitory effect of heparin on the expression of TF induced by various mitogenic (FCS, PDGF-BB and EGF) and non-mitogenic (bacterial LPS) agents. Inhibition by heparin of SMCs proliferation induced by the same agonists was also determined. Quiescent human vascular SMCs from normal adult arteries were treated for 1 h by heparin and related sulfated polysaccharides before stimulation by the agonists. All the agonists up-regulated the expression of TF antigen and activity. TF expression induced by the growth factors was inhibited by heparin (IC 50: 10-30 microg/ml), and other sulfated polysaccharides (IC 50: 1-5 microg/ml). SMCs proliferation, late activation of the extracellular signal-regulated kinases (ERK1/2), and PKC activity were inhibited by heparin (IC 50: 30-50 microg/ml) in SMCs stimulated by FCS but not in SMCs treated by PDGF or EGF. In contrast, heparin had no effect on LPS-induced TF expression nor on LPS-induced PKC activation. These results indicate that, besides its well known effect on SMC proliferation, heparin displays an inhibitory effect on cell mediated blood clotting processes through regulation of the TF expression.     

Anticoagulant effects of an antidiabetic drug on monocytes in vitro

Introduction

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.

Methods

We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).

Results

Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.

Conclusions

Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.     

Relevance of tissue factor and tissue factor pathway inhibitor for hypercoagulable state in the lungs of patients with idiopathic pulmonary fibrosis

We investigated the role of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Bronchoalveolar lavage (BAL) fluid was obtained from 22 patients with IPF, and the levels of TF and TFPI antigen were measured by ELISA. The TF and TFPI levels in BAL fluid supernatant were significantly higher in IPF patients than in normal controls. In addition, both levels were significantly higher in advanced cases than in nonadvanced cases. There was a significant correlation between the TF and TFPI levels. Localization of TF and TFPI antigens was investigated by immunohistochemical staining. Both antigens were mainly localized in hyperplastic cuboidal epithelial cells, suggesting that the widespread distribution of these cells contributed to the increase of TF and TFPI antigen levels in the lungs of IPF patients. To assess whether TF activity is counterbalanced by TFPI in the lungs of IPF patients, we examined procoagulant activity and TF activity. It was found, however, that both procoagulant and TF activities were significantly higher in the BAL fluid supernatant of IPF patients than in that of normal controls, which suggested that TFPI was actually increased, but the increase was insufficient to counterbalance TF, leading to the development of a hypercoagulable state in the lungs of IPF patients.     

Tissue factor pathway inhibitor reduces experimental lung metastasis of B16 melanoma

The importance of tissue factor (TF) in tumor biology has been highlighted by studies suggesting its involvement in cell signaling, metastasis and angiogenesis. Since many animal studies have shown that anticoagulant therapy can reduce experimental metastasis, we studied whether the natural inhibitor of TF-mediated blood coagulation, Tissue Factor Pathway Inhibitor (TFPI), might be similarly effective. Using a murine experimental model, we found that intravenous injection of recombinant murine TFPI immediately before introduction of tumor cells reduced metastasis by 83% (P < 0.001). B16 murine melanoma cells stably transfected with a TFPI expression vector exhibited reduced lung seeding following intravenous injection by 81% (P < 0.001) compared with controls. No difference in primary tumor growth was observed between TFPI+ and control cells. Mice receiving intravenous somatic gene transfer of sense TFPI expression vector developed 78% fewer lung nodules than controls (P < 0.05). We conclude that TFPI has significant anti-metastatic activity in this experimental model.     

Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk

Introduction

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.

Methods

TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.

Results

TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p < 0.001). TF protein expression was also increased in endometroid carcinoma (P < 0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P < 0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P < 0.01). There was no difference in TFPI expression between the two groups.

Conclusion

TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.     

New observations on procoagulant properties of amniotic fluid: Microparticles (MPs) and tissue factor-bearing MPs (MPs-TF), comparison with maternal blood plasma

Introduction

Microparticles (MPs) are submicron fragments of the cell membrane affecting many biological processes, e.g. coagulation. The aim of the study was to determine (i) MPs and (ii) tissue factor bearing MPs (MPs-TF) in the amniotic fluid and in blood plasma of parturient women, as well as to assess (iii) TF and TFPI.

Material and methods

The study group consisted of 38 women laboring at term, whereas the control group included 20 non-pregnant women. ELISA method was used to evaluate MPs, MPs-TF, TF and TFPI.

Results

The levels of MPs and MPs-TF were significantly higher in the amniotic fluid than in blood plasma of parturient women: the level of MPs was 41.08 times higher (medians: 246.48 nM PS vs. 6.00 nM PS, respectively, p < 0.001), and the level of MPs-TF was 18.59 times higher (medians: 90.16 pg/ml vs. 4.85 pg/ml, respectively) (p < 0.001).

Conclusions

1. Microparticles (MPs) and tissue factor-bearing MPs (MPs-TF) are constituent components of amniotic fluid. 2. It is reasonable to assume that these components together with tissue factor (TF) and its inhibitor (TFPI) can participate in life-threatening coagulation disturbances in amniotic fluid embolism, and to take into consideration their impact on fetal development.