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Cardiovascular Research 2006 69(1):218-226; doi:10.1016/j.cardiores.2005.07.013
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Copyright © 2005, European Society of Cardiology

Indobufen inhibits tissue factor in human monocytes through a thromboxane-mediated mechanism

Sonia Eliginia, Francesco Violic, Cristina Banfib, Silvia S. Barbieria, Marta Brambillab, Mirella Saliolac, Elena Tremolia,b and Susanna Collia,*

aE. Grossi Paoletti Center, Department of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy
bDepartment of Cardiac Surgery, Centro Cardiologico Fondazione Monzino I.R.C.C.S, University of Milan, Italy
cDivisione IV Clinica Medica, Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza", Policlinico Umberto I, Rome, Italy

* Corresponding author. Tel.: +39 02 50319913; fax: +39 02 50318250. Email address: Susanna.Colli{at}unimi.it

Received 9 February 2005; revised 13 June 2005; accepted 14 July 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To assess whether indobufen, a reversible inhibitor of platelet cyclooxygenase (Cox) activity, affects tissue factor (TF) in human monocytes and to investigate the relationship between Cox-derived products and TF.

Methods: TF was evaluated in isolated adherent monocytes, both resting and lipopolysaccharide (LPS)-stimulated, in terms of procoagulant activity, protein, and mRNA levels. The expression of TF surface antigen was determined in LPS-stimulated whole blood monocytes by flow cytometry. The levels of the stable thromboxane A2 (TxA2) metabolite, TxB2, and of prostaglandin E2 (PGE2) were measured in monocyte supernatant by immunoenzymatic techniques. Cox-1 and Cox-2 protein level, tyrosine phosphorylation, and mitogen-activated protein kinase (MAP-kinase) activation were determined by Western blot analysis.

Results: Indobufen prevents TF expression and activity both in isolated and in whole blood monocytes. Reduction of TxA2 synthesis, coupled with a lack of effect on PGE2 levels and prevention of ERK1/2 phosphorylation are highlighted as the mechanisms through which indobufen negatively affects TF.

Conclusions: Data show that indobufen down-regulates TF in monocytes. This novel activity, coupled with the antiplatelet effect of the drug, may add benefit for its use in the management of atherothrombosis.

KEYWORDS Indobufen; Tissue factor; Thromboxane; Monocytes; Thrombosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Indobufen is a reversible inhibitor of platelet cyclooxygenase (Cox) activity, and, as a consequence, it suppresses thromboxane A2 (TxA2) synthesis [1–3]. It is effective in a broad spectrum of prothrombotic conditions as graft occlusion after CABG surgery [4–6], restenosis after carotid endarterectomy [7], thromboembolic events in patients with heart disease [8], and intermittent claudication [9]. Interestingly, in the latter condition, the effect of indobufen has been attributed to an impairment of neutrophil activation through Cox inhibition [10]. Moreover, indobufen possesses the same efficacy as warfarin in the secondary prevention of thromboembolic events in at risk patients with non-rheumatic atrial fibrillation [11]. In addition, the ability of indobufen to suppress enhanced TxA2 synthesis in episodes of platelet activation during the acute phase of unstable angina, has been recently highlighted [12]. This effect, which is not shared by aspirin, has been attributed to the inhibition of Cox-2, which is expressed by monocytes in response to a local inflammatory milieu.

Tissue factor (TF), a transmembrane glycoprotein of the cytokine receptor superfamily, is the cellular receptor and cofactor for plasma factor VIIa. The binary complex TF/factor VIIa proteolytically activates factor IX and X and triggers the coagulation pathway that, ultimately, leads to the generation of thrombin and fibrin [13,14]. TF represents the main initiator of thrombogenesis in vivo [15]. Monocytes synthesize and express TF in response to a variety of agents. Increased monocyte TF activity is implicated in thrombotic complications associated with acute and chronic inflammatory disease [16]. TF also plays a crucial role in atherosclerosis, which represents a chronic low level inflammatory disease [17]. The pivotal contribution of TF to thrombosis, occlusion of coronary vessels, and to myocardial infarction, has been extensively documented and points to this protein as a possible therapeutic target [18–21]. TF levels are heightened in several disease states as unstable angina, unfavorably related to the outcomes of the disease [22–24], and myocardial infarction [25]. Moreover, elevated monocyte TF in CABG patients [26,27] and in post-angioplasty may contribute to occlusion and re-occlusion of coronary vessels [28].

In this study we show that indobufen prevents TF expression in both isolated and whole blood monocytes. Reduction of TxA2 synthesis and prevention of ERK1/2 phosphorylation are among the mechanisms underlying drug effect.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Reagents
Racemic indobufen (2-[p(1-oxo-isoindolin-2-yl)phenyl]-butyric acid) was from Pharmacia, Milano, Italy. Lipopolysaccharide (LPS, from E. coli 0114:B4) was from Difco Labs., Detroit, MI, USA. IBOP, furegrelate, arachidonic acid, NS-398, SQ-29548, acetylsalicylic acid, bovine serum albumin (fatty acid-free and low endotoxin), octyl-β-D-glycopyranoside were from Sigma Chemical Co., Milano, Italy. Prostaglandin E2 (PGE2), SC-58125 and prostaglandin endoperoxide H2 (PGH2) were from Cayman Chemical (Spi-Bio, Massy Cedex, France). PD98059, SB203580, jun N-terminal kinase (JNK) inhibitor I (L-form, cell permeable) and its negative control were from Calbiochem (Inalco SpA, Milano, Italy). Electrophoresis reagents were from Amersham Pharmacia Biotech., Milano, Italy. Cell culture medium and reagents were purchased from BioWhittaker Italia SRL, Bergamo, Italy.

2.2 Antibodies
Monoclonal antibodies against TF (mAb 9-5B7) were a gift from J. H. Morrissey (University of Illinois, Urbana-Champaign, USA) and from Y. Nemerson (Mount Sinai School of Medicine, New York, USA). Monoclonal antibodies against cyclooxygenases (Cox-2, mAb 29 and Cox-1, mAb 10 and 11) were gifts from A. Habib (American University of Beirut, Lebanon). Antibody against phosphotyrosine was from Upstate Biotechnology (D.B.A., Milano, Italy). Monoclonal antibodies against total and phosphorylated forms of ERK1/2 and p38 were from Cell Signaling Inc., (Celbio S.r.l, Milano, Italy) and Biosource Inc., (Prodotti Gianni S.p.A, Milano, Italy), respectively. Peroxidase-conjugated anti-mouse IgG antibody was from Jackson ImmunoResearch Labs Inc. (Li StarFISH, Milano, Italy).

2.3 Cell isolation and stimulation
Monocytes were isolated from blood of healthy donors. Blood sampling was performed in accordance with the principles outlined in the Declaration of Helsinki. Mononuclear leukocytes were separated by centrifugation on a Ficoll–Paque density gradient and monocytes were isolated by selective adherence (90 min) to tissue culture dishes [29]. Cell preparations were >90% monocytes, as determined by non-specific esterase staining. Unless otherwise specified, adherent monocytes were incubated with various agents in medium M-199 supplemented with 0.5% antibiotics, 1% glutamine, and 2% human AB serum for 1 h, and then exposed to fresh medium containing LPS (0.4 ng/ml) for a further 3 h. Incubation time and LPS concentration were selected on the basis of results obtained in preliminary experiments. The endotoxin content of all culture materials and reagents was measured by the Limulus amebocyte lysate assay (BioWhittaker) and only those containing <3 pg/ml endotoxin were used.

2.4 Evaluation of TF activity
Monocytes were lysed with 15 mM octyl-β-D-glycopyranoside and TF activity was evaluated as procoagulant activity measured in cell lysates by the one-step plasma recalcification time assay [30]. Clotting times were converted into arbitrary TF units using a reference curve obtained with a standard human TF preparation (Thromborel S, Behring, Marburg, Germany) containing 106 U/ml. The logarithm of clotting time was related to the logarithm of TF activity. The specificity of the assay was confirmed by the absence of TF activity in cells preincubated with a mouse monoclonal antibody against human TF (American Diagnostica Inc, Greenwich, CT, USA). Experiments performed with factor VII deficient plasma (Stago Boheringer, Milan, Italy) verified that reductions in clotting times were attributable to TF. Data are expressed as units (U) of TF activity/µg cell protein.

2.5 Western blot analysis
Cells were harvested in lysis buffer, pH 6.8, as described [31]. Cell debris was removed by centrifugation (10,000 x g for 5 min) and protein concentration was determined by the micro-bicinchoninic acid assay. Equal amounts of lysates were subjected to SDS-PAGE and transblotted onto nitro-cellulose membrane by a semidry transfer unit (Hoefer Scientific Instruments). Membranes were incubated for 1 h with antibodies directed against TF (1 µg/ml), Cox-1 (5 µg/ml), Cox-2 (1/10,000), ERK1/2 (1/2000) and p38 (1/1000), total and phosphorylated. Phosphotyrosine detection was performed on membranes stripped and reprobed with an antibody against phosphotyrosine (1/5000). Blots were incubated with peroxidase-conjugated secondary antibody (1/5000) for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech.). Optical density was assessed using Multianalyst software (Bio-Rad Labs).

2.6 RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted from cells by TRIzol Reagent (Invitrogen Life Technologies Italia SRL, Milan, Italy). TF mRNA levels were detected by a coupled reverse transcription-polymerase chain reaction (RT-PCR). Oligonucleotides from the coding sequence of the human TF gene were the following: 5'-ACTACTGTTTCAGTGTTCAAGGAGTGATTC-3' (a 30-mer sense, corresponding to nucleotides 722–751) and 5'-ATTCAGTGGGGAGTTCTCCTTCCAGCTCTG-3' (a 30-mer antisense, corresponding to nucleotides 925–954). Primers were also synthesized to amplify the cDNA encoding GAPDH (5'-CCACCCATGGCAAATTCCATGGCA-3', a 24-mer sense oligonucleotide at position 216, and 5'-TCTAGACGGCAGGTCAGGTCCACC-3', a 24-mer antisense oligonucleotide at position 809. Oligonucleotides were purchased from Invitrogen Life Technologies. Total cellular RNA from each sample (0.5 µg) was reverse transcribed using a thermal cycler GeneAmp PCR System 2400 (Perkin Elmer Life Science, Wallac Italia, Milan, Italy) [31]. Amplified products were resolved by electrophoresis through a 2% agarose gel and detected after staining with ethidium bromide.

2.7 Determination of thromboxane B2 (TxB2) and PGE2 release by adherent monocytes
Cox-2 activity was determined in adherent monocytes either unstimulated or exposed to LPS for 3 h. Cells were washed in Hank's buffer, pH 7.4, containing 1 mg/ml bovine serum albumin and incubated for 30 min with 10 µM arachidonic acid or 1 µM PGH2 in the same buffer. Levels of TxB2, the stable inactive TxA2 metabolite, and of PGE2 were measured by enzyme immunoassay (EIA, Cayman Chemical, Spi-Bio, Massy, Cedex, France) in monocyte supernatants.

2.8 Whole blood stimulation and flow cytometry
Blood from healthy volunteers was drawn into Vacutainer tubes containing lithium heparin. Aliquots of blood were placed into sterile polystyrene tubes and incubated for 1 h with indobufen. After this time, LPS (0.4 ng/ml) was added and the blood was further incubated for 3 h. At the end of incubation, samples were fixed with 1% formaldehyde for 30 min at 4 °C and subsequently stained with phycoerythrin-conjugated antibody anti-CD14 (Becton Dickinson) and fluorescein isothiocyanate antibody anti-TF (American Diagnostica). Isotype control monoclonal antibodies were used in each experiment to determine non-specific background. The percentage of TF-positive events within this population, determined as having a stronger fluorescence than the uppermost 2% of cells stained with isotypic control, was calculated. 3000 CD14-positive events were counted on each sample.

2.9 Statistical analysis
Data, reflecting measurements in monocytes from different donors, are reported as mean ± S.E. Paired observations were compared using Student's t test. Grouped differences were compared with ANOVA (Fisher's LSD test). p ≤ 0.05 values were considered statistically different. Statistical analysis was performed with Sigma Stat.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Indobufen prevents TF in isolated and whole blood monocytes
Adherent monocytes possess TF activity, which increased upon incubation with LPS (0.4 ng/ml). Indobufen significantly prevented LPS-induced TF activity that was reduced almost to basal levels by 250 µM concentration (Fig. 1, panel A). The same effect was also observed in whole blood monocytes exposed to LPS (Fig. 1, panel B). The reduction of TF antigen in CD14-positive monocyte population was 32 ± 15% and 55 ± 12%, by 100 and 250 µM indobufen, respectively, mean ± S.E., p<0.05 vs. LPS, n=4).


Figure 1
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  		Fig. 1 Indobufen prevents TF in LPS-stimulated monocytes both isolated and in whole blood. (A) Adherent monocytes were incubated with indobufen for 1 h. LPS was then added for 3 h. TF activity was determined in triplicate using the one-step clotting assay. Data are the mean ± S.E. from 8 experiments performed with monocytes isolated from different donors. §p<0.001 vs. unstimulated condition; *p<0.01 vs. LPS. (B) Whole blood was incubated with indobufen and LPS as above. TF was detected by flow cytometry. Data (mean ± S.E.) from 4 experiments performed with blood isolated from different donors. *p<0.05 vs. LPS-stimulated whole blood monocytes. (C, D) Adherent monocytes were incubated with indobufen for 1 h prior to stimulation with LPS for 3 and 1 h, respectively. TF protein was determined by Western blotting. TF mRNA levels were determined by RT-PCR. GAPDH mRNA was used as internal standard. The sequences of the primers for TF and GAPDH are given under Methods. Result is representative of 3 independent experiments giving similar results.

 
Inhibition of TF activity was attributable to reduced levels of TF protein and mRNA (Fig. 1, panel C and D). TF activity of resting monocytes was also reduced by indobufen (from 15.18 ± 3.53 to 7.83 ± 1.84 TF units/µg protein upon incubation with 250 µM indobufen, mean ± S.E., p<0.05, n=7).

3.2 Role of Cox-1 and Cox-2 activity in TF induction
Acetylsalicylic acid (ASA), at a concentration that selectively inhibits Cox-1 (100 µM) did not affect TF activity. In contrast, TF was prevented by the selective Cox-2 inhibitors NS-398 [32] (Fig. 2) and SC-58125 (5 µM) [33] (data not shown). Higher ASA concentration (10 mM), reported to inhibit both Cox-1 and Cox-2 activity [34], reduced TF by 61.15 ± 26.7%, mean ± S.E, n=6.


Figure 2
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  		Fig. 2 Effect of Cox-1 and Cox-2 inhibitors on TF activity in human adherent monocytes stimulated with LPS. Monocytes were incubated with ASA or NS-398 for 1 h. LPS was then added for 3 h. TF activity was determined in triplicate using the one-step clotting assay. Data are the mean ± S.E. from 10 experiments performed with monocytes isolated from different donors. §p<0.001 and *p<0.05 vs. unstimulated and LPS-stimulated monocytes, respectively.

 
No difference in cell viability was observed at any dose of the tested drugs, as assessed by Neutral Red.

3.3 Indobufen prevents TxA2 synthesis in LPS-stimulated monocytes
Exposure of monocytes to LPS induced Cox-2 expression, while Cox-1 protein levels were unaltered (Fig. 3). Increase of Cox-2 levels was associated with enhanced levels of TxB2 and PGE2 in monocyte supernatant (Fig. 4, panel A and B).


Figure 3
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  		Fig. 3 Indobufen does not affect Cox-1 and Cox-2 protein. Monocytes were incubated with indobufen for 1 h prior to stimulation with LPS for 3 h. Cox-1 and Cox-2 protein levels were determined by Western blot. Blots are representative of 3 separate experiments performed with monocytes from different donors.

 

Figure 4
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  		Fig. 4 Indobufen reduces TxB2 in a concentration-dependent manner without affecting PGE2 levels in human adherent monocytes stimulated by LPS. (A, B) Monocytes were incubated with indobufen for 1 h prior to LPS addition. Incubation was continued for 3 h. TxB2 and PGE2 levels were measured by enzyme immunoassay in monocyte incubation medium after the addition of arachidonic acid (10 µM). Data (mean ± S.E.) from 5 experiments performed with monocytes isolated from different donors. §p<0.005, #p<0.05 vs. unstimulated monocytes; *p<0.05 vs. LPS-stimulated monocytes. Indobufen does not affect TxA2 synthase in monocytes. (C) Monocytes were incubated with indobufen for 1 h prior to LPS addition for 3 h. TxA2 synthase was assessed by Western analysis. Blot is representative of 3 independent experiments giving similar results.

 
Indobufen did not affect both Cox-1 and Cox-2 protein (Fig. 3), whereas it reduced TxB2 levels (Fig. 4, panel A). In contrast, PGE2 levels were not influenced (Fig. 4, panel B). The reduction of TxB2 levels was not dependent on changes of TxA2 synthase enzyme levels (Fig. 4, panel C) or activity, measured in supernatants after monocyte incubation with 1 µM PGH2 (data not shown).

3.4 Opposite regulation of TF by TxA2 and PGE2 in human monocytes
To gain more insight into the mechanism of action of indobufen, a set of experiments were carried out aimed at evaluating the role of TxA2 and its receptor (TP) in LPS-induced monocyte TF. Monocytes were incubated for 1 h with the TxA2 synthase inhibitor furegrelate or with the TP antagonist SQ-29548. Cells were then exposed to LPS for 3 h. LPS-induced TF activity was significantly reduced by both furegrelate and SQ-29548 (Fig. 5, panel A), indicating that TxA2 synthesis and the interaction with its receptor represent key events in LPS-induced TF. This notion was reinforced by results from experiments carried out with the TxA2 mimetic IBOP which abrogated the effect of indobufen (Fig. 5, panel B). Moreover, IBOP was able to induce TF protein in adherent monocytes per se (Fig. 5, panel C). This effect was attributable to increased tyrosine phosphorylation of several cellular proteins with molecular masses of approximately 73, 82, 89, and 118 kDa. The pattern of phosphorylation observed with IBOP was comparable to that induced by LPS (Fig. 6). Pretreatment of adherent monocytes (1 h) with the tyrosine kinase inhibitor herbymicin (1 µg/ml) resulted in reduction of TF activity (not shown). This finding is in accordance with previous data obtained in murine macrophages [35].


Figure 5
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  		Fig. 5 TxA2 increases TF in human adherent monocytes stimulated with LPS. (A, B) Monocytes were incubated with furegrelate or SQ-29548 or indobufen or IBOP for 1 h prior to LPS addition for 3 h. TF activity was determined in triplicate using the one-step clotting assay. Data (mean ± S.E.) from 5 experiments performed with monocytes isolated from different donors. §p<0.01 vs. unstimulated monocytes; *p<0.05 vs. LPS-stimulated monocytes. (C) Monocytes were incubated with IBOP for 3 h. TF protein was determined by Western analysis. Blot is representative of at least 3 independent experiments.

 

Figure 6
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  		Fig. 6 IBOP and LPS increase protein tyrosine phosphorylation in human adherent monocytes. Monocytes were incubated with LPS or IBOP for 3 h. Protein tyrosine phosphorylation was determined by Western analysis. Blot is representative of at least 3 independent experiments.

 
In contrast to TxA2 that acts positively on TF induction, PGE2 prevented TF activity of monocytes, both resting and LPS-stimulated (Fig. 7).


Figure 7
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  		Fig. 7 PGE2 reduces TF activity in human adherent monocytes. Monocytes were incubated with PGE2 for 1 h prior to LPS addition for 3 h. TF activity was determined in triplicate using the one-step clotting assay. Data (mean ± S.E.) from 4 experiments performed with monocytes isolated from different donors. §p<0.01 vs. unstimulated monocytes; *p<0.05 vs. LPS-stimulated monocytes.

 
3.5 Effect of indobufen on MAP-kinase activation in human monocytes
Preliminary experiments were performed to address the role of MAP-kinase activation in LPS-mediated TF induction. Cells were preincubated for 30 min with PD98059 (25 µM), SB203580 (10 µM) or the JNK inhibitor peptide I (1 µM), that inhibit ERK1/2, p38 and JNK, respectively. Monocytes were subsequently exposed to LPS for further 3 h. LPS-induced TF activity was prevented by PD98059 and SB203580 (–56.66 ± 6.56%, mean ± S.E, n=3, and –80.25 ± 5.82%, mean ± S.E, n=4, respectively), but not by the JNK inhibitor peptide. This finding points to a role of ERK1/2 and p38 MAP-kinase in TF induction by LPS. The effect of indobufen on ERK1/2 and p38 phosphorylation was then evaluated by Western blot analysis. LPS increased the levels of phosphorylated ERK1/2 and p38 with an onset at 10 min. Values were still above the basal level until 40 min (Fig. 8, panels A and B). Indobufen reduced the extent of ERK1/2 phosphorylation (Fig. 8, panel A), whereas the levels of phosphorylated p38 were unaltered (Fig. 8, panel B). Overall, these data indicates that ERK1/2 blockade is involved in TF inhibition by indobufen in LPS-treated monocytes.


Figure 8
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  		Fig. 8 Indobufen reduces the extent of ERK1/2 phosphorylation induced by LPS in human adherent monocytes. (A, B) Monocytes were incubated with or without indobufen for 1 h prior to LPS addition. Cell lysates were analyzed by Western analysis for phosphorylated and total ERK1/2 and p38 kinases (A and B). The result shown is representative of those from 3 independent experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major finding of the present study is that indobufen dose-dependently down-regulates TF in LPS-stimulated monocytes both isolated and in whole blood.

Inhibition of TxA2 derived from Cox-2 activity and prevention of ERK1/2 activation are responsible for TF reduction. The effect occurs at drug concentrations reached in plasma after repeated oral administration at conventional doses [36,37].

Another relevant finding is that Cox-2 metabolites are key regulators of TF induction in monocytes. Firstly, results obtained with inhibitors of Cox-1 and Cox-2 indicate that inhibition of Cox-2 activity results in reduced TF activity. Secondly, we show that Cox-2-derived TxA2 is involved in TF induction through the interaction with its receptor. TPs have been characterized in human peripheral blood monocytes ({alpha} isoform, TP{alpha}) [38] and the central role of TPs in the regulation of LPS-induced TF activity in human whole blood has been recently delineated ex vivo [39].

Our data indicate that indobufen inhibits TxA2 but not PGE2 synthesis in LPS-stimulated monocytes. Of interest, TxA2 and PGE2 play an opposite effect in the regulation of TF; results obtained with inhibitors of TP or TxA2 synthase indicate that TxA2 positively regulates TF induction by LPS, whereas PGE2 negatively affects it.

This opposite effects of PGE2 and TxA2 in the modulation of TF is similar to that reported in peripheral blood mononuclear cells exposed to arachidonic acid for 20 h [40]. In addition, the ability of the TxA2 mimetic IBOP to counteract TF inhibition by indobufen reinforces the finding of the positive role played by TxA2 in TF induction by LPS.

In our condition, TxA2 is synthesized in a larger amount to PGE2 in monocytes, either resting or exposed to LPS. This finding indicates that, at least in the time frame adopted in our study, TxA2 is the Cox-2 metabolite that prevails, depending upon differences in both kinetic and induction profile of the terminal synthases in response to inflammatory agents [41–43].

A role of the 5-lipoxygenase metabolite leukotriene B4 as amplifier of TF induction in whole blood exposed to LPS has been reported [44]. Of interest, indobufen, at concentration similar to that used in our study, reduces leukotriene B4 formation in whole blood incubated with calcium ionophore A23187 [GenBank] [45].

The link between TxA2 and TF has been recognized in pathology. Increased synthesis of thromboxane and increased procoagulant activity have been detected in arachidonic acid challenged monocytes from type 2 diabetic patients comparative to controls [46].

Tyrosine phosphorylation is among the earliest signaling events involved in macrophage TF expression in response to proinflammatory agents [35]. Our data show that LPS increases phosphotyrosine accumulation in monocytes, an effect that persisted until 3 h. Of note, the TxA2 mimetic IBOP increased tyrosine phosphorylation with a pattern fully comparable to that of LPS. This finding indicates that TxA2 and LPS share similarity in early signaling pathways leading to TF expression. Interestingly, it has been shown that IBOP phosphorylates TP in A7r5 cells [47].

The role of MAP-kinase in LPS-induced TF in monocytic cells has been appreciated only recently [48,49]. Our results obtained with the MAPK inhibitors SB203580 and PD98059 clearly show that both ERK1/2 and p38 activation play a major role in TF transcription by LPS in human monocytes. Indobufen prevented ERK1/2 activation and this effect may give reason for the reduced TxA2 levels released by indobufen-treated monocytes. Interestingly, it has been recently reported that TxA2 induces ERK phosphorylation via its receptors in several cell types [50–52].

Indobufen inhibits TF activity also in unstimulated adherent monocytes. It has been shown that TF expression in monocytes is integrin-dependent and that β2-integrin signaling in monocytic cells flows through tyrosine phosphorylation and activation of ERK1/2, which is essential for the subsequent TF expression [53]. Moreover, the adherence primes human monocytes in terms of TxA2 release [54].

Of relevance, the effect of indobufen on TF was also observed in whole blood monocytes exposed to LPS, an experimental condition characterized by the cooperation between monocytes and other cells/cell microparticles in terms of TF induction [55].

Our data is of major interest in view of the recent observation of a beneficial effect of anti-TF therapy in myocardial ischemia–reperfusion injury in the rabbit [56]. In the cited study, the reduction of the infarct size observed after TF inhibition by an anti-TF antibody, indicates that anti-TF therapy should be of significant clinical benefit in the treatment of acute coronary syndromes. The inflammatory component that accompanies this clinical condition suggests that investigative efforts should be directed toward the development of effective inhibitors of local and systemic TF and inflammation as well. The beneficial effect may, indeed, stem both from inhibition of thrombin and from reduction of inflammation. Indobufen, by the concomitant inhibition of TF activity and prostanoid formation, represents therefore a good candidate drug to control the hypercoagulable state which goes in parallel with inflammation in various clinical settings, ranging from acute lung injury [57] to sepsis [58].


   Acknowledgements
 
This work was supported by grants from the Italian Ministry of University and Scientific Research and University of Milan (FIRB 2001-RBNE01BNFK and FIRST 2003, grant to S. C.). The authors wish to thank Eduardo Stragliotto (Medical Department, Pfizer Italia, Rome, Italy) for the constant encouragement. The excellent technical assistance of Ms. Nadia Marinoni is gratefully acknowledged.


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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