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The Journal of Immunology, 1998, 160: 6145-6150.
Copyright © 1998 by The American Association of Immunologists

Arachidonic Acid Enhances the Tissue Factor Expression of Mononuclear Cells by the Cyclo-Oxygenase-1 Pathway: Beneficial Effect of n-3 Fatty Acids1

Yves Cadroy2, Dominique Dupouy and Bernard Boneu

Laboratoire de Recherche sur l’Hémostase et la Thrombose, Centre Hospitalo-Universitaire Purpan, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocytes express tissue factor (TF) upon stimulation by inflammatory agents. Dietary administration of fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in an impairment of TF expression by monocytes. EPA and DHA are metabolized differently from arachidonic acid (AA), the major fatty acid present in cell membranes. We examined the effects of AA on the TF expression of isolated human PBMC, and we determined whether EPA and DHA modulated this phenomenon differently. Nonstimulated PBMC had a low TF-dependent procoagulant activity. When PBMC were incubated with increasing concentrations of AA, the TF-dependent procoagulant activity increased in a dose-dependent manner to 190% at 7.5 µM. Indomethacin, a cyclo-oxygenase inhibitor, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 or of 5-lipoxygenase had no inhibitory effect. A thromboxane (TX)A2/endoperoxides receptor antagonist and a TX synthase inhibitor blocked the potentiating effect of AA. Purified PGG2 and carbocyclic TXA2, a TXA2 agonist, enhanced the procoagulant activity of PBMC in a dose-dependent manner whereas, in contrast, PGE2 inhibited it. Finally, contrary to AA, EPA or DHA did not increase TXB2 production or TF expression by PBMC. The TF-dependent procoagulant activity of isolated PBMC was increased by AA through the production of cyclo-oxygenase-1 metabolites; the combined action of PGG2 and TXA2, which potentiated it, was greater than that of PGE2, which inhibited it. Dietary n-3 fatty acids exert part of their beneficial effect by modulating this procoagulant activity differently from AA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Arachidonic acid (AA;3 20:4, n-6) is a polyunsaturated fatty acid present in cell membranes that modulates diverse physiologic and pathologic responses. These effects are primarily mediated by its metabolic products, eicosanoids. Eicosanoids include notably the cyclo-oxygenase products, i.e., PG and TX, and the lipoxygenase products, i.e., leukotrienes and hydroxyeicosatetraenoic acids (HETEs). Their effects, which depend on the nature of compounds, principally concern inflammation and thromboregulation (1). Eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) are other polyunsaturated fatty acids contained in fish oil. EPA is metabolized into eicosanoids that are different from the corresponding compounds derived from AA and are considered to develop reduced biologic activities (2, 3, 4, 5). DHA is not metabolized by cyclo-oxygenase or lipoxygenase enzymes (6). Experimental (7, 8), epidemiologic, and interventional (9, 10) studies suggest that consumption of fish oil, which causes a progressive decrease in the AA content of tissue phospholipids and an increase in the tissue level of n-3 fatty acids, exerts beneficial effects on thrombosis and atherosclerosis. The molecular mechanisms by which dietary n-3 fatty acids exert their beneficial effects are complex and have not been totally clarified. Some of these effects have been attributed to the activity of n-3 fatty acids on monocytes (4, 8, 11, 12, 13).

Monocytes are centrally involved in numerous pathophysiologic processes, such as thrombosis, atherosclerosis, wound repair, and inflammation. These properties are partly related to their ability to express various procoagulant activities (14, 15, 16, 17). The procoagulant activities of monocytes are mediated to a large extent by cell surface-associated tissue factor (TF). TF is the cellular receptor and cofactor for plasma factor VII(a), which initiates the coagulation protease cascade leading ultimately to the generation of thrombin and fibrin (18). Dietary administration of fish oil results in an impairment of TF expression by monocytes (8, 12, 13). The molecular mechanisms underlying TF activity reduction after n-3 fatty acids intake are unknown. However, since monocytes are important generators of eicosanoids and possess both cyclo-oxygenase and lipoxygenase enzymes, changes in the monocyte capacity to generate eicosanoids by these pathways have been suspected to influence monocyte TF activity. Thus, several recent studies have indicated that eicosanoids may modulate the procoagulant properties of monocytes. For example, prostacyclin analogues and PGE1 cause a decrease in cytokine-induced TF activity by monocytes (19, 20), whereas, conversely, platelet 12-HETE enhances PBMC procoagulant activity (21).

To clarify the mechanisms of the beneficial effect of dietary fish oil on the procoagulant activity of monocytes, we investigated in vitro the effects of AA, EPA, and DHA on TF expression by isolated PBMC. We show for the first time that AA, but not EPA or DHA, enhanced the TF-dependent procoagulant activity of PBMC. The roles of the cyclo-oxygenase and lipoxygenase pathways were also examined. We describe a novel role for the cyclo-oxygenase-1 pathway in modulating the TF expression of AA-stimulated PBMC that appeared to be balanced by the opposite effects of PGE2, which inhibited it, and endoperoxides/TXA2, which potentiated it.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and cell cultures

PBMC were isolated using a modification of the method described by Balter et al. to efficiently deplete platelet from the cell preparation (22). Whole blood was obtained with a 19-gauge needle from healthy volunteers who had not taken aspirin or other nonsteroidal anti-inflammatory drugs in the 7 days preceding the donation. Blood was anticoagulated with trisodium citrate (0.129 M; Becton Dickinson, Meylan, France) and centrifuged at 280 x g for 15 min at 4°C. Platelet-rich plasma was removed. The sedimented cells were diluted to twice the original blood volume with PBS (pH 7.4; Seromed, Biochrom, Berlin, Germany), layered onto Ficoll-Hypaque PLUS (Pharmacia Biotech, Uppsala, Sweden), and centrifuged at 400 x g for 35 min at 4°C. The resulting PBMC were washed in 5 mM EDTA-PBS four to six times to remove remaining platelets The resulting mononuclear fraction contained less than two platelets per leukocyte. Nonspecific {alpha}-naphtyl-acetate esterase staining indicated that the mononuclear fraction contained 28.3 ± 6.4% (n = 4) monocytes. Cells (500 µl) were incubated at 2.5 x 106 PBMC/ml in medium 199 (ATGC Biotechnologie, Noisy-le-Grand, France) containing 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in stoppered, sterile, pyrogen-free tubes.

Sodium arachidonate, eicosapentaenoate, and docosahexaenoate (Sigma, Saint Quentin Fallavier, France), stored under argon at -80°C, or carbocyclic TXA2 (CTXA2; Sigma), TXB2 (Cayman, Ann Arbor, MI), PGG2, E2, D2, and F2{alpha} (Calbiochem, La Jolla, CA) were dissolved in medium 199 just before use and added to the cell suspension at the concentrations indicated below for 20 h. Indomethacin (10 µM; Sigma), NS398 (1 µM; Calbiochem), furegrelate (10 µM; Cayman), baicalein (10 µM; Calbiochem), or L655,238 (1 µM; Calbiochem), selective inhibitors of cyclo-oxygenase, cyclo-oxygenase-2, TX synthase, 12-lipoxygenase, and 5-lipoxygenase, respectively, and SQ29,548 (10 µM; Cayman), a TXA2/endoperoxides receptor antagonist, were added before adding sodium arachidonate to the cells. The concentration used for each of these inhibitors was chosen based on the IC50 reported by the respective manufacturers. Depending on the reagents, they were dissolved in water, ethanol, or DMSO and added (1 µl) to the cells. Control experiments performed with the respective solvents indicated that they did not affect the procoagulant activity of PBMC (data not shown). LPS was obtained from Escherichia coli 0111:B4 (Sigma) and incubated with PBMC for 20 h in some experiments (see below).

Human endothelial cells were isolated from umbilical veins and cultured according to the method of Jaffe et al. (23). The culture medium was composed of RPMI 1640 and medium 199 (ATGC Biotechnologie, Noisy-Le-Grand, France) supplemented with 20% human pooled serum (Institut Jacques Boy, Reims, France). The cells were identified by their typical morphology. Cells were incubated for 4 h in medium 199 and carefully scraped with a rubber policeman just before measurement of their procoagulant activity.

Cell viability, as assessed by the measurement of lactate dehydrogenase release in the supernatant of cultured cells and by trypan blue exclusion, was >90%. All reagents used for cell isolation and culture were prepared with endotoxin-free water. The level of endotoxin contamination in the different reagents incubated with PBMC, as assessed by a sensitive chromogenic Limulus assay (Chromogenix, Molndal, Sweden), was very low (<0.001 ng/ml, final concentration). This level of endotoxin did not enhance the procoagulant activity of PBMC (data not shown).

Measurement of procoagulant activity

Procoagulant activity was measured on intact cells using a one-step plasma recalcification time assay performed on a coagulometer (KC4, Amelung, Lemgo, Germany). The PBMC were placed on ice for 10 to 20 min to remove any adherent monocytes. A 20-µl sample of PBMC or scrape-harvested endothelial cells was added to 90 µl of citrated normal human platelet-poor plasma. One hundred microliters of 25 mmol/l CaCl2 was added to initiate the reaction. Coagulation times were converted into procoagulant activity units (AU) using reference curves determined with a standard human brain TF preparation containing 106 AU/ml (Thromborel S, Behring, Marburg, Germany); the logarithm of the procoagulant activity was related to the logarithm of the coagulation time. The procoagulant activity of mononuclear cells was characterized by incubating the cells with a mixture of two mouse anti-human TF mAbs (10 µg/ml; American Diagnostica, Greenwich, CT) for 30 min at 37°C.

Determination of TF, TXB2, and PGE2

TF Ag was measured on cell lysates by commercially available ELISA (Imubind Tissue Factor, American Diagnostica). Cell lysates were prepared by lysing PBMC in PBS containing 1% Triton X-100, 1 mM EDTA (Merck, Chelles, France), 16 mM octyl PD glucopyranoside (Boehringer Mannheim, Meylan, France), 10 µM pepstatin A, 10 µM leupeptin, 0.1 mM PMSF (Sigma), and 100 kallkrein inhibitor units/ml aprotinin (Sanofi-Choay, Paris, France). The cell lysates were then frozen and thawed three times. They were stored at -80°C until assayed. TXB2 and PGE2 produced by cultured PBMC were measured in the supernatant by enzyme immunoassays (Cayman).

Statistical analysis

All results represent the mean ± 1 SEM of 4 to 10 separate experiments. Depending on the data, statistical analysis was performed using Student’s test for paired variables or ANOVA followed by a Neuman-Keuls test when p <= 0.05. Differences were considered significant at p <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AA enhanced the procoagulant activity and TF synthesis of mononuclear cells

Nonstimulated PBMC had a very low procoagulant activity (138 ± 17 AU/106 cells; n = 7). Considering that the procoagulant activity of the cell suspension was solely due to monocytes, which represented 28% of the PBMC suspension, the procoagulant activity expressed by monocytes (493 ± 61 AU/106 monocytes) was comparable to that expressed by cultured resting endothelial cells (498 ± 110 AU/106 endothelial cells; n = 9), indicating that monocytes were not significantly stimulated by the procedure of isolation. The procoagulant activity was TF dependent, since it was inhibited >90% by preincubating the cells with anti-TF mAbs. When PBMC cells were incubated with AA (5 µM) for 4 and 20 h, respectively, their procoagulant activity increased, but this increase was less marked with 4-h incubation (i.e., by 132% at 4 h vs 335% at 20 h; n = 1). Therefore, all additional experiments were performed with 20-h incubation. In these conditions, AA increased the procoagulant activity in a dose-dependent manner (Table IGo) to a mean of 190% at 7.5 µM (n = 10), which corresponds to a mean reduction in clotting times of 40 s (from 240 to 200 s). Higher concentrations resulted in cell lysis. Similarly, AA significantly enhanced the cell content of TF Ag (Table IIGo). By comparison, LPS (100 ng/ml) increased the procoagulant activity of PBMC by 294% (406 ± 50 AU/106 cells; n = 7).


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  		Table I. Effect of AA and of selective inhibitors of the cyclooxygenase and lipoxygenase pathways on the procoagulant activity of PBMC1

 

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  		Table II. Effect of AA, EPA, and DHA on TF Ag synthesis and TXB2 production by human mononuclear cells1

 
The effect of AA was mediated by the cyclo-oxygenase-1 pathways

Preincubation of PBMC with indomethacin (10 µM), a cyclo-oxygenase-1 and -2 inhibitor, which blocked TXA2 formation by 96 ± 1%, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 (NS398) or 5-lipoxygenase (L655,238) had no inhibitory effect (Table IGo). Importantly, the stimulating effect of AA was not due to a low contamination by endotoxins, since the procoagulant activity induced by LPS remained unchanged in the presence of indomethacin (563 ± 123 vs 570 ± 88 AU/106 cells with and without indomethacin, respectively; n = 8). Therefore, these results indicate that AA enhanced the procoagulant activity of PBMC by the cyclo-oxygenase-1 pathway.

PGG2 and CTXA2 enhanced the procoagulant activity and TF synthesis of PBMC

The major metabolites of AA produced by the cyclo-oxygenase-1 pathway are represented by the endoperoxides PGG2/PGH2 and further metabolized into TXA2, PGE2, PGD2, and PGF2{alpha}. To determine those that are responsible for the enhancing effect of AA on the procoagulant activity of PBMC, we tested the effect of purified PG (Table IIIGo). PGG2 and CTXA2, a TXA2 agonist, enhanced, in a dose-dependent manner, the procoagulant activity of PBMC and TF Ag (from 0.32 ± 0.12 to 1.15 ± 0.33 ng/106 cells at 2 µM PGG2; p < 0.05; n = 8). In contrast, PGE2 significantly decreased the procoagulant activity of the cells. TXB2, PGD2, and PGF2{alpha} had no significant effect.


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  		Table III. Effect of purified prostaglandins on the procoagulant activity of mononuclear cells1

 
To further test the hypothesis that the formation of endoperoxides and TXA2 was involved in the potentiating effect of AA on the procoagulant activity of PBMC, we studied the effect of furegrelate, a TX synthase inhibitor, which blocked TXA2 formation by 94 ± 1%, and that of SQ29,548, a TXA2/endoperoxides receptor antagonist. Both molecules inhibited the potentiating effect of AA on the procoagulant activity of PBMC (Table IGo). As expected, the procoagulant effect of CTXA2 was not inhibited by furegrelate (266 ± 51 vs 142 ± 30 AU/106 cells with and without CTXA2; 1 µM; p < 0.01; n = 4).

The potentiating effect of AA on the procoagulant activity of PBMC and the respective amounts of TXB2, the stable metabolite of TXA2, and PGE2 released by PBMC varied among the different blood donors from whom the cells were isolated. The effect of AA on the procoagulant activity of cells was not directly related to the amount of TXB2 or PGE2 released (p > 0.20), but was significantly and positively related to the ratio of TXB2/PGE2 formed by cells in presence of AA (r = 0.61; p < 0.001; Fig. 1Go). This correlation remained significant when the outlying data with the highest procoagulant activity (937% of the baseline procoagulant activity) were deleted (r = 0.44; p < 0.01). Therefore, the variability of response of blood donors to AA appeared to be related to the respective amount of endoperoxides/TXA2 and PGE2 produced by PBMC; there was a balance between these eicosanoids, which showed opposite effects on the procoagulant activity of PBMC.



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  		FIGURE 1. Relationship between the procoagulant activity and the molar ratio of TXB2/PGE2 produced by PBMC isolated from different blood donors and incubated with AA. PBMC (2.5 x 106 cells/ml) isolated from different blood donors were incubated without or with AA (5 µM) for 20 h. The procoagulant activity with 5 µM AA was expressed as a percentage of the baseline procoagulant activity obtained in the absence of AA. TXB2 and PGE2 were measured in the supernatant of PBMC (n = 35).

 
To confirm that the procoagulant activity of PBMC was determined in part by a balance between TXA2 and PGE2, PBMC were incubated with PGE2 (0.5 µM) and increasing concentrations of CTXA2 (0 to 1 µM). As found previously (Table IIIGo), the addition of PGE2 to PBMC decreased their baseline procoagulant activity by 54% (Fig. 2Go). When the molar ratio CTXA2/PGE2 was increased from 0 to 2, the procoagulant activity of PBMC increased in a ratio-dependent manner. However, although the increase in procoagulant activity produced by the addition of CTXA2 to PGE2-treated cells was statistically significant (p < 0.01), the procoagulant activity of PGE2-treated cells remained significantly lower (p < 0.05) than that of cells incubated with CTXA2 but without PGE2 (p < 0.05).



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  		FIGURE 2. Effects of increasing concentrations of CTXA2 on the procoagulant activity of PBMC, treated or not with PGE2 (n = 5). PBMC (2.5 x 106 cells/ml) were incubated for 20 h with PGE2 and CTXA2. Results are expressed as a percentage of the procoagulant activity of resting PBMC incubated for 20 h without PGE2 or CTXA2. Statistical comparisons of the procoagulant activity of PBMC incubated with purified PGE2 and CTXA2 vs that obtained without PGs are represented by asterisks (* indicates p < 0.05; ** indicates p < 0.01).

 
EPA and DHA did not enhance the procoagulant activity and TF synthesis of mononuclear cells

The n-3 fatty acids EPA and DHA are metabolized differently from AA by cyclo-oxygenase and lipoxygenase enzymes. For example, when these fatty acids were incubated with PBMC, EPA, unlike AA, did not increase the basal production of TXB2, and DHA inhibited it (Table IIGo). In these conditions, EPA and DHA did not enhance the procoagulant activity (Fig. 3Go) and TF Ag level (Table IIGo) of PBMC. At 5 and 7.5 µM, the procoagulant activity was significantly lower with EPA and DHA (p = 0.01) than with AA. Therefore, these results confirm that the effect of AA was mostly mediated by the production of endoperoxides and TXA2.



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  		FIGURE 3. Effects of AA, EPA, and DHA on the procoagulant activity of PBMC (n = 9). PBMC (2.5 x 106 cells/ml) were incubated for 20 h with increasing concentrations of AA, EPA, or DHA. Statistical comparisons of the procoagulant activity of PBMC incubated in presence of fatty acids vs that obtained without fatty acids are represented by asterisks (** indicates p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in the present study that the procoagulant activity of isolated PBMC was enhanced by AA. This increase in procoagulant activity was due to an increased generation of TF and not to an increased surface expression of procoagulant activity (Table IIGo). The effect of AA was mediated through the production of cyclo-oxygenase-1 metabolites, especially endoperoxides and TXA2. These findings differ from those of a previous study in which AA enhanced the procoagulant activity of PBMC when they were stimulated by endotoxins and in presence of platelets. This result was related to the platelet lipoxygenase product, 12-HETE (21). The role of this platelet lipoxygenase product was negligible in our study, since the platelet/PBMC ratio was low (i.e., less than two platelets per leukocyte) and since baicalein, a pharmacologic inhibitor of platelet 12-lipoxygenase, did not inhibit the effect of AA (Table IGo). Our study was performed with nonstimulated PBMC. AA and other reagents were not contaminated with LPS; no detectable amount of endotoxin was found in the various molecules incubated with the cells, and the effect of AA was inhibited with indomethacin, contrary to that induced by LPS.

AA is metabolized by PBMC through different enzymatic pathways, notably cyclo-oxygenase-1, cyclo-oxygenase-2, and 5- and 12-lipoxygenases (22, 24, 25, 26). Leukotrienes, which are important modulators of the inflammatory response (27), were not involved in the enhancing effect of AA on the procoagulant activity of nonstimulated PBMC, since it was not affected by 5- and 12-lipoxygenase inhibitors (L655,238 and baicalein, respectively; Table IGo). In addition, previous studies have shown that leukotriene B4, produced by the 5-lipoxygenase pathway, has no effect on the procoagulant activity of PBMC (21, 28). 12-HETE is a potent cofactor for TF generation by PBMC (21). However, in our working conditions, the amount of 12-HETE released may have been too low to have a role, since there were very few platelets, an important source of 12-HETE, and since monocytes produce only very low amounts of 12-HETE, especially in the absence of LPS (26).

The enhancing effect of AA observed in our study was mediated by cyclo-oxygenase-1. It was inhibited by indomethacin, a cyclo-oxygenase-1 and -2 inhibitor (29), but it remained unchanged with a selective inhibitor of cyclo-oxygenase-2 (NS398; Table IGo). The latter result is not surprising, since the cyclo-oxygenase-2 pathway is only functional when monocytes are exposed to inflammatory stimuli, which were absent in our study (24, 25). Interestingly, previous works and our own preliminary data indicate that pathways other than cyclo-oxygenase-1 are involved in the enhancing effect of AA on the procoagulant activity of PBMC when they are stimulated with LPS (21, 30).

Our experiments were performed with exogenous AA. However, we do consider the role of cyclo-oxygenase-1 pathway in AA-induced TF expression to be pathophysiologically relevant. Indeed, this polyunsaturated fatty acid was effective at concentrations as low as 5 µM. The plasma concentrations of polyunsaturated fatty acids have been reported to vary between 0.2 and 2 mM (31), and the percentage of AA in plasma free fatty acid can reach 9% (32). Interestingly, and in contrast to AA, endotoxin-stimulated TF expression of PBMC was not inhibited by inhibitors of the cyclo-oxygenase or lipoxygenase pathway. Thus, endogenous AA, present in the cell membrane, and/or the cyclo-oxygenase or lipoxygenase pathways did not appear to be involved in endotoxin-induced TF expression.

In the presence of AA, monocytes release endoperoxides (PGG2 and PGH2) through the cyclo-oxygenase-1 pathway that are further metabolized into two major metabolites, TXA2 and PGE2 (22, 24, 25). Purified PGG2 and CTXA2, a TXA2 agonist, enhanced the TF expression of the cells in a dose-dependent manner, whereas, in contrast and as previously indicated, purified PG of the E series decreased the procoagulant activity of PBMC (19, 20) (Table IIIGo). In addition, furegrelate, a TX synthase inhibitor, and SQ29,548, a TXA2/endoperoxide receptor antagonist, abolished the enhancing effect of AA on the procoagulant activity of PBMC (Table IGo). Furthermore, our results suggest that there is a balance between the respective anti- and procoagulant effects of PGE2 and endoperoxides/TXA2. The variability of response of PBMC from different blood donors to AA was positively related to the respective amounts of TXB2 and PGE2 released by the cells (Fig. 1Go). Similarly, a recent study showed that levels of cytokine production (i.e., TNF-{alpha} and IL-1ß) in human monocytes were determined in part by the balance between TXA2 and PGE2 production (33).

We showed that in vitro EPA and DHA, unlike AA, did not enhance the TF-dependent procoagulant activity of PBMC (Fig. 3Go). Similarly, administration of diets rich in n-3 fatty acids to animals or humans results in a reduction of TF activity by monocytes (8, 12, 13). Our results indicate that this effect is related to changes in the cyclo-oxygenase metabolites produced in the presence of fatty acids contained in fish oil compared with those produced by AA. When fish oils are fed to humans, a substantial amount of the n-3 polyunsaturated fatty acids, EPA and DHA, accumulates in cell membrane phospholipids, and the AA content decreases (3). Thus, the reduction of TF activity by n-3 fatty acids is not immediate and appears well correlated to changes in the fatty acid composition of cell membranes (13). With n-3 fatty acids, lower levels of TXA2, a procoagulant mediator, are produced (3). EPA is converted by cyclo-oxygenase to the 3-series TX and PG (2, 3). PGH3 and TXA3 are relatively inactive with regard to platelet aggregation and are synthesized in small amounts, since EPA is a relatively poor substrate for cyclo-oxygenase. DHA is not metabolized by cyclo-oxygenase and directly inhibits this enzyme (6), as suggested by the results presented in Table IIGo.

This study is the first to show that endoperoxides and TXA2 enhance the TF-dependent procoagulant activity of PBMC. Previous works have indicated that PGE2 decreases endotoxin-induced TF synthesis in human monocytes by elevating cAMP through stimulation of adenylate cyclase (34). Therefore, we suggest that endoperoxides/TXA2 enhanced TF expression of PBMC by inhibiting adenylate cyclase, lowering cAMP, and inducing a rise in the concentration of ionized calcium in the cell cytoplasm (1). Our study adds one more mechanism by which TXA2 may be prothrombotic. Other mechanisms include its capacity to cause platelet aggregation and vasoconstriction, to augment monocyte and neutrophil adhesiveness (1, 35, 36, 37, 38), and to promote diapedesis (3). Finally, our results also suggest that part of the antithrombotic effect of aspirin, which irreversibly inactivates cyclo-oxygenase and inhibits the production of endoperoxides/TXA2, may be mediated by a diminution of the procoagulant activity of PBMC.


   Footnotes
 
1 This work was supported in part by a grant from Pierre Fabre, Castres, France. Back

2 Address correspondence and reprint requests to Dr. Yves Cadroy, Laboratoire d’Hématologie, Pavillon Lefèbvre, Centre Hospitalo-Universitaire Purpan, 31059 Toulouse Cedex, France. Back

3 Abbreviations used in this paper: AA, arachidonic acid; TX, thromboxane; HETE, hydroxyeicosatetraenoic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; TF, tissue factor; CTXA2, carbocyclic thromboxane A2; AU, procoagulant activity units. Back

Received for publication October 3, 1997. Accepted for publication February 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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