Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer

Urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-I, play a key role in tumor invasion and metastasis. They were the first novel tumor biological factors to be validated at the highest level of evidence (LOE I) regarding their clinical utility in breast cancer. Their antigen levels are determined in tumor tissue extracts by standardized, quality-assured immunometric assays (ELISA). Since the late 1980s, numerous independent studies have demonstrated that patients with low levels of uPA and PAI-I in their primary tumor tissue have a significantly better survival than patients with high levels of either factor. These prognostic data have recently been validated by an EORTC (European Organization for Research and Treatment of Cancer) pooled analysis comprising more than 8,000 breast cancer patients. In addition, results from a multicenter prospective randomized therapy trial in node-negative breast cancer ("Chemo N(0)") showed that node-negative breast cancer patients with low levels of uPA and PAI-I in their primary tumor have a very good prognosis, and may thus be candidates for being spared the burden of adjuvant chemotherapy. In contrast, node-negative patients with high uPA/PAI-I are at substantially increased risk of disease recurrence, comparable to that of patients with three or more tumor cell positive axillary lymph nodes. The "Chemo N(0)" trial as well as retrospective data also indicate that these high-risk patients benefit from adjuvant chemotherapy. In conclusion, over a period of about 15 years sufficient evidence has been put forward to demonstrate that determination of uPA and PAI-I in primary breast cancer patients supports risk-adapted individualized therapy decisions, particularily in patients with node-negative disease.     

Increase in levels of plasminogen activator and type-1 plasminogen activator inhibitor in human breast cancer: possible roles in tumor progression and metastasis

We measured antigen levels of two kinds of plasminogen activators, tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (UK), as well as their primary inhibitor, type-1 plasminogen activator inhibitor (PAI-1) in the tissue extracts of benign and malignant breast tumors. Tumor tissues of 36 fibroadenomas and 39 breast cancers were examined. t-PA levels were not different in both groups. Malignant tumors contained the significantly higher levels of UK than benign tumors (p less than 0.001). Furthermore in breast cancer tissues, UK antigen levels of tumors with axillary lymph node involvements were significantly higher than those of tumors without lymph node involvements (p less than 0.05). PAI-1 antigen levels of breast cancer tissues were dramatically higher than those of fibroadenoma (p less than 0.001). PAI-1 levels of node positive carcinomas showed also values significantly higher than node negative ones (p less than 0.01). When we divided cancer tissues into three groups as node negative tumors, tumors with positive axillary nodes fewer than four and tumors with four or more positive nodes, PAI-1 levels increased corresponding to the progression of lymph node involvements (p less than 0.05). Immunohistochemical studies, using mouse monoclonal antibodies to human UK and PAI-1, showed that those immunoreactivities were diffusely distributed in the cytoplasm of human breast cancer cells. Their staining patterns were very similar to each other.     

The tissue factor and plasminogen activator inhibitor type-1 response in pediatric sepsis-induced multiple organ failure

STUDY OBJECTIVE: Cytokines increase endothelial tissue factor (TF) and tissue plasminogen activator inhibitor type-1 (PAI-1) expression in vitro. Tissue factor interacts with factor VII to facilitate thrombosis and PAI-1 inhibits fibrinolysis by endogenous plasminogen activators. Because cytokine release is increased in children with sepsis-induced multiple organ failure (MOF), we hypothesized a cytokine associated increase in circulating TF and PAI-1 antigen release, and systemic activity in these patients. STUDY DESIGN: One hundred and seven consecutive children, who met the criteria for sepsis, and 10 critically ill children without sepsis, were enrolled in the study. Plasma TF and PAI-1 antigen and activity levels, Interleukin-6 antigen levels (IL-6), nitrite + nitrate levels (marker of nitric oxide production) and number of organs failing were measured on days 1-3 of sepsis. RESULTS: Increased TF and PAI-1 antigen, and PAI-1 activity levels were associated with increasing IL-6 and nitrite + nitrate levels (p <0.05), the development of MOF (p <0.05), and mortality (p <0.05). Increased systemic PAI-1 activity was associated with cardiovascular, renal. and hepatic failure (p <0.05). Increased systemic TF activity was associated with the development of coagulopathy (p <0.05) and tended to be associated with mortality (p = 0.06, power .77) CONCLUSIONS: A shift to an anti-fibrinolytic endothelium phenotype characterizes children who develop sepsis-induced MOF and mortality. Children with coagulopathy have a shift to a pro-coagulant phenotype. These findings support potential therapeutic roles for PAI-1 and TF pathway inhibitors in reversal of this devastating pathophysiologic process.     

Ceramide-enhanced urokinase-type plasminogen activator (uPA) release is mediated by protein kinase C in cultured microglia

As described previously, a relatively high dose of neurotrophins increased the release of urokinase-type plasminogen activator (uPA) from cultured microglia. This biological response is suggested to be caused by ceramide, which is a metabolite of nerve growth factor low-affinity receptor (NGFRp75)-associated sphingomyelin turnover. Therefore, in the present study, we examined the effect of ceramide on the release of uPA from cultured microglia. Treatment of the cells with permeable C8-ceramide (D-erythro-Sphingosine, N-octanoyl-) enhanced uPA release in a dose-dependent manner. This effect of C8-ceramide was mimicked by treatment with bacterial sphingomyelinase. A pharmacological study using a specific PKC activator, phorbol-12-myristate-13-acetate, and a protein kinase C (PKC) inhibitor, bisindolylmaleimide, showed that PKC activation is required in order to release uPA from ceramide-stimulated microglia as well as from nonstimulated microglia. Further study using a specific conventional PKC (cPKC) activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and a specific cPKC inhibitor, G? 6976, suggested that PKC-delta and/or -epsilon is involved in uPA release. As opposed to the apoptotic pathway, however, no activation of c-Jun N-terminal kinase and nuclear factor kappa B was observed in C8-ceramide-stimulated microglia. The findings suggest that uPA release from microglia is regulated by a mechanism in which PKC-delta and/or -epsilon are activated and further signals are transduced subsequently.     

Plasminogen activator inhibitor type-1 (PAI-1) and its role in cardiovascular disease

Cardiovascular disease is responsible for approximately 50% of total mortality in Europe, the USA and Japan. Established risk factors including smoking, hypercholesterolemia, and hypertension explain about half of the incidence of cardiovascular disease only. Reduced endogenous fibrinolytic activity secondary to increased plasma activity of plasminogen activator inhibitor type-1 (PAI-1) is now considered as a new cardiovascular risk factor. In this review, evidence is gathered for the notion that PAI-1 constitutes a predictor of cardiovascular disease and also contributes to the development of cardiovascular disease as a pathogenetic factor. The review will focus on experimental studies modulating PAI-1 activity and clinical studies addressing coronary heart disease, myocardial infarction, restenosis after coronary angioplasty, and graft occlusion after coronary artery bypass grafting.     

The urokinase-type plasminogen activator/its soluble receptor system is independently related to carotid atherosclerosis and associated with CC-chemokines in uraemic patients

INTRODUCTION: The urokinase-type plasminogen activator (uPA)/its soluble receptor (suPAR) and CC-chemokines are two systems contributing in the atherosclerosis. We try to establish whether these two systems were associated among themselves, and the significance of this potential association in relation to carotid atherosclerosis in uraemic patients. MATERIALS AND METHODS: We determined uPA, suPAR, CC-chemokines: monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory proteins (CCL3, CCL4), regulated upon activation, normal T cell expressed and secreted (CCL5) and the intima-media thickness (IMT) values in uraemic patients on peritoneal dialysis (CAPD), haemodialysis (HD) and healthy controls. RESULTS: The values of the uPA/suPAR system, CCL2, CCL4 and IMT in the patients significantly exceeded those in controls, whereas CCL5 were lower in the patients than in the controls. CCL3 concentrations were similar in all investigated groups. CCL2, CCL4 and CCL5 were positively associated with uPA/suPAR system. CCL2 and CCL4 were associated with IMT. Multivariate analysis showed that uPA, suPAR and age were the strong independent variables linked to IMT values. CONCLUSIONS: The carotid atherosclerosis is independently related to uPA/its soluble receptor system in dialysis patients. In addition, the strong positive associations existed between uPA/suPAR system and CC-chemokines suggesting that these two systems could cooperate and influence of atherosclerosis in these patients.     

Prognostic value of pre-surgical plasma PAI-1 (plasminogen activator inhibitor-1) levels in breast cancer

Introduction

Plasminogen activator inhibitor (PAI-1) may have an independent prognostic value in breast cancer (BC). PAI-1 4G/5G polymorphism may have significance for antigen expression. Thus, we analyzed the possible associations between PAI-1 4G/5G polymorphism, plasma PAI-1 levels, and clinicopathological features of breast cancer (BC) patients.

Patients and Methods

PAI-1 4G/5G polymorphism (both on germinal and tumor DNA) and plasma PAI-1 levels were investigated in 99 BC patients and 50 unrelated healthy women similar for age and menopausal status.

Results

No association was found between allele frequencies and clinicopathological features of BC or plasma antigen levels. Plasma PAI-1 levels were higher in BC compared to controls (p = 0.002), particularly in patients with large tumors (p < 0.001). 5-year follow-up was achieved in 79 patients: 30% had relapsing disease, 63% with positive compared to 37% with negative PAI-1 levels (p < 0.05). 5-year relapse-free survival rate of positive PAI-1 was 46% vs., 77% of negative patients (p = 0.02).

Conclusions

We may conclude that plasma PAI-1 levels in BC patients could represent a useful prognostic variable for relapse, although PAI-1 polymorphism might not represent a genetic susceptibility factor.     

Importance of plasminogen activator inhibitor type 1 (PAI-1) for preventing single chain urokinase plasminogen activator (scu-PA) conversion into two chain urokinase plasminogen activator (tcu-PA) in plasma in vitro.

We have studied the effects of PAI-1 on the conversion of scu-PA into tcu-PA in vitro in plasma containing or not a 125I-fibrin clot by determining tcu-PA activity on S2444. Two preparations of PAI-1 have been used, a fraction of medium conditioned with the monkey Vero cells (Vero-Prep), the antiurokinase activity of which is inhibited at 83% by anti PAI-1 IgG, or purified human PAI-1 from HT 1080 fibrosarcoma cells. Scu-PA purified from human kidney cells has been treated with diisopropylfluorophosphate before use. In plasma, conversion of scu-PA into the tc form is accelerated by addition of anti PAI-1 IgG. In plasma containing a clot, generation of tcu-PA, is considerably delayed after addition of the Vero-Prep or human PAI-1. Clot lysis is also decreased but to a lesser extent than it would be expected from the level of tcu-PA activity. Addition of anti PAI-1 antibodies shortens the lag phase before tcu-PA appears and moderatly accelerates clot lysis. These results demonstrate the importance of PAI-1 for the stability of scu-PA in plasma in vitro by delaying its conversion into tcu-PA.     

Absence of synergism between tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) and urokinase on clot lysis in a plasma milieu in vitro

A potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in citrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and alpha 2-antiplasmin. t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant alpha 2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal. Combinations of t-PA and scu-PA, of t-PA and urokinase or of scu-PA and urokinase at thrombolytic doses of each showed no synergism for thrombolysis. Fifty percent clot lysis in 2 h was obtained at total concentrations of the combined agents of 5 to 15 nM with molar ratios ranging from 1:4 to 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Independence of growth factor induced plasminogen activator inhibitor type-1 (PAI-1) expression from the 4G/5G polymorphism of the PAI-1 gene

Increased PAI-1 expression has been implicated in accelerating atherogenesis. Increases under some conditions are modulated by growth factors. Genetic factors such as the 4G/5G polymorphism in the promoter of the PAI-1 gene play a role under certain circumstances. The present study was designed to delineate for the first time interactions between growth factors and the 4G/5G polymorphism with respect to PAI-1 expression in human arterial smooth muscle cells (HASMC). HASMC were genotyped and exposed to growth factors. PAI-1 gene and protein expression were induced consistently by TGF-beta (up to 4.0-fold), PDGF (2.1-fold), TNF-alpha (1.7-fold), and thrombin (2.3-fold). Results were similar regardless of which genotype (4G/4G [n=9], 4G/5G [n=13], and 5G/5G [n=7]) was present. The induction of increased PAI-1 expression in human arterial smooth muscle cells by growth factors implicated in accelerated atherogenesis is independent of the PAI-1 4G/5G polymorphism. Accordingly, modulation of PAI-1 expression is likely to be influenced predominantly by environmental factors acting on, rather than genetic factors intrinsic to the PAI-1 promoter.     

Renal and hepatic handling of endogenous tissue-type plasminogen activator (t-PA) and its inhibitor in man

In eight male patients with normal liver and kidney function fibrinolytic components were measured in arterial blood and in renal and hepatic vein blood, obtained during catheterization for analysis of hypertension. Blood samples were collected simultaneously from veins und corresponding arteries before and 5 minutes after the completion of intravenous injection of desmopressin (DDAVP), 0.4 micrograms/kg body weight over a 10 minute period. DDAVP induced a rise in t-PA antigen and activity, and in von Willebrand factor, accompanied by a decrease in free PA-inhibitor level. We failed to detect a significant rise in plasma urokinase activity. The concentrations of fibrinogen, plasminogen, alpha 2-antiplasmin, antithrombin III and coeruloplasmin did not change either. Renal production of t-PA under basal conditions was inferred from a negative arterio-venous (A-V) difference in t-PA-activity and in t-PA-antigen levels but this could not be confirmed by orthogonal regression analysis of the same data. A-V differences of other fibrinolytic factors were negligible. In the hepatic vessels a significant positive A-V difference of t-PA-activity and of t-PA-antigen levels was a uniform finding. After DDAVP, when plasma levels were elevated, the mean A-V difference was proportionally higher, consistent with a constant fractional elimination rate. Free PA-inhibitor was virtually absent from arterial blood after DDAVP, but appeared in hepatic vein blood, indicating either production of the inhibitor by the liver or dissociation of a circulating complex of t-PA and its inhibitor in the liver. The blood levels of the other investigated components did not show any change upon passage through the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Daytime fluctuations of plasminogen activator inhibitor 1 (PAI-1) in populations with high PAI-1 levels.

The mechanism underlying diurnal variations in PAI-1 as well as the cellular origin of PAI-1 in subjects with high PAI-1 levels are unknown. We evaluated diurnal changes (8:00 am vs 4:00 pm) in PAI-1 (functional and immunological assays), t-PA Ag and t-PA/PAI-1 complex levels in controls and subjects with high PAI-1 levels. Three test groups were recruited among obese hyperinsulinemic subjects, emergency care unit patients with inflammatory syndrome or infection and pregnant women. The classical afternoon decrease of PAI-1 level was observed in controls and obese subjects but its amplitude was greater in the latter. The decrease in t-PA Ag and t-PA/PAI-1 complex levels was the same in controls and in obese. As, in previous studies, elevated PAI-1 levels have been correlated with insulin resistance and a decrease in insulin sensibility has been described in the early morning, it is proposed that this "dawn phenomenon" could be implicated in the circadian variations of PAI-1 in controls and could be amplified in obese subjects. Great variability in PAI-1, t-PA Ag or t-PA/PAI-1 complex levels was observed in patients with acute inflammatory syndrome or infection for whom classical biorhythms are suppressed. No diurnal changes in PAI-1 and other fibrinolytic parameters were observed in patients with inflammatory syndrome or in pregnant women suggesting that other sources and/or other regulatory mechanisms of PAI-1 production are involved.     

血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性研究

目的探索PAI及TAFI对急性脑梗死静脉溶栓后出血性转化的预警作用。方法前瞻性的将溶栓患者分为出血转化组和无出血转化组,对两组患者溶栓前及溶栓后静脉血浆PAI及TAFI浓度进行对比及统计学分析。结果溶栓前两组基线PAI-1水平差异有明显统计学意义(P=0.037)。溶栓后次日复查晨血PAI-1水平出血转化组明显较无出血转化组值更低(P=0.035)。溶栓前出血转化组的基线TAFI水平较无出血转化组比较TAFI值更低(P=0.024)。溶栓后次日复查晨血出血转化组TAFI值同样低于无出血转化组(P=0.042)。结论血浆中PAI-1和TAFI水平与溶栓后出血性转化相关性较高。当溶栓前及溶栓后PAI-1及TAFI的低水平表达均可增加溶栓后出血性转化的风险。     

An enzyme-linked immunosorbent assay (ELISA) used to study the cellular secretion of endothelial plasminogen activator inhibitor (PAI-1)

A two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml-1 sample. PAI-1 was detected in primate sera but not in a wide range of non-primate sera and no cross-reactivity with alpha 2-antiplasmin or antithrombin III was observed. The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for approximately equal to 10% of total secreted protein. Specific activity of intracellular PAI-1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t1/2 for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.     

The resistance of fibrin-stimulated tissue plasminogen activator to inactivation by a class pai-2 inhibitor (minactivin)

Solid phase fibrin was an efficient stimulator of the tissue-type plasminogen activator (t-PA), and the plasmin produced could be detected by colorimetric assay of the soluble phase above the fibrin. However the fibrin-stimulated activity of t-PA was not inhibited by minactivin. This result was in contrast to that obtained with poly-D-lysine (PL) stimulated t-PA, where minactivin was a potent inhibitor. However, if PL was added to fibrin-bound t-PA, the enzyme once again became susceptible to minactivin inhibition. This occurred without release of t-PA from the fibrin matrix. Minactivin alone did not bind to fibrin or to the t-PA fibrin complex. It was therefore concluded that minactivin normally has no significant role in the regulation of t-PA mediated fibrinolysis, but this effect can be induced by PL.     

Expression of the urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) in brain tissues of human immunodeficiency virus patients with opportunistic cerebral diseases

The urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) play an important role in cell migration and extracellular proteolysis. We previously described uPAR/uPA overexpression in the cerebrospinal fluid (CSF) and brain tissues of patients with human immunodeficiency virus (HIV)-related cerebral diseases. In this study, we examined uPAR/uPA expression by immunohistochemistry (IHC) in brains of HIV patients with opportunistic cerebral lesions and in HIV-positive/negative controls. uPAR was found in macrophages/microglia with the highest levels in cytomegalo-virus (CMV) encephalitis, toxoplasmosis, and lymphomas; in cryptococcosis and progressive multifocal leukoencephalopathy (PML) cases, only a few positive cells were found and no positivity was observed in controls. uPA expression was demonstrated only in a few macrophages/microglia and lymphocytes in all the cases and HIV-positive controls without different pattern of distribution; no uPA immunostaining was found in cryptococcosis and HIV-negative controls. The higher expression of uPAR/uPA in most of the opportunistic cerebral lesions supports their role in these diseases, suggesting their contribution to tissue injury.     

Progesterone regulation of plasminogen activator inhibitor 1 (PAI-1) antigen and mRNA levels in human endometrial stromal cells.

Plasminogen activator activity decreases in the endometrium in the secretory phase of the menstrual cycle. This is partly due to decreased release of urokinase plasminogen activator in response to progesterone. Plasminogen activator inhibitor type 1 (PAI-1) is an efficient inhibitor of both tissue-type and urokinase-type plasminogen activators, and may therefore be instrumental for the control of plasminogen activation. In this study we examined the effects of steroid hormones on PAI-1 release and PAI-1 mRNA levels in primary cultures of human endometrial stromal cells. In these cells the secretion of PAI-1 was increased by progesterone in a dose and time dependent way, but was not affected by estradiol. The progesterone induction of PAI-1 secretion was preceded by a 7-8 fold increase of the steady state level of PAI-1 mRNA in the cells, suggesting that progesterone activates PAI-1 gene expression. Cultured endometrial glandular epithelial cells were found to release only insignificant amounts of PAI-1 with or without hormone treatment. The effect of progesterone on endometrial stromal cells was mimicked by DH-testosterone. However, while the response to progesterone was completely blocked by ZK112993, a potent antagonist of the progesterone receptor, the response to DH-testosterone was partially blocked by ZK112993, and partially by OH-flutamide, a potent antagonist of the androgen receptor. This suggests that a secretory response on PAI-1 expression is mediated via androgen receptors in endometrial tissue.     

Relationship between lipoprotein(a) phenotypes and plaminogen activator inhibitor type 1 in diabetic patients

It has been demonstrated in vitro that lipoprotein(a) [Lp(a)] increases the endothelial synthesis of plasminogen activator inhibitor 1 (PAI-1). However, this effect in vivo is controversial, and the possible relationship between PAI-1 and Lp(a) phenotypes has not been evaluated. The aim of the study was to determine the influence of Lp(a) and its phenotypes on PAI-1 serum concentrations in diabetic patients. For this purpose we include 75 Caucasian diabetic patients (34 consecutive type I and 41 consecutive type II) without late diabetic complications. Lp(a) and PAI-1 were assessed by ELISA. Lp(a) phenotypes were determined by SDS-PAGE followed by immunoblotting, and grouped according to size in small (F,B,S1,S2), big (S3,S4), and null. A linear correlation between Lp(a) and PAI-1 was not observed either as a whole or when type I and type II diabetic patients were analyzed separately. However, significant differences were detected in PAI-1 levels when Lp(a) phenotypes were considered (small: 42.1+/-31.8 ng/mL; big: 37.2+/-26.1 ng/mL; null: 14.4+/-14.4; p< 0.05). The significant differences were due to the low PAI-1 concentrations observed in patients with null phenotype. Our results suggest that fibrinolytic activity might be preserved in diabetic patients with null Lp(a) phenotype. Furthermore, it could be speculated that diabetic patients with null phenotype should be considered at low risk to develop cardiovascular disease.     

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

Introduction

Heme 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 Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment 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.

Conclusions

CORM-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.