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CARDIOVASCULAR

Antiplatelet Activity of J78 (2-Chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), an Antithrombotic Agent, Is Mediated by Thromboxane (TX) A₂ Receptor Blockade with TXA₂ Synthase Inhibition and Suppression of Cytosolic Ca²+ Mobilization

College of Pharmacy, Chungbuk National University, Cheongju, Korea (Y.-R.J., M.-R.C., J.-T.H., K.-S.L., J.-J.L., Y.L., Y.-P.Y.); College of Pharmacy, Ewha Women's University, Seoul, Korea (C.-K.R.); College of Pharmacy, Seoul National University, Seoul, Korea (J.-H.C.); and Research Center for Bioresource and Health, Chungbuk National University, Cheongju, Korea (D.-Y.Y., M.-Y.L., Y.-P.Y.)

Received July 7, 2004; accepted August 17, 2004.

	Abstract

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We previously reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, exhibited a potent antithrombotic effect, which might be due to antiplatelet rather than anticoagulation activity. In the present study, possible anti-platelet mechanism of J78 was investigated. J78 concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 µg/ml), thrombin (0.05 U/ml), arachidonic acid (100 µM), and U46619 [GenBank] (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F₂; 1 µM), a thromboxane (TX) A₂ mimic, with IC₅₀ values of 0.32 ± 0.01, 0.44 ± 0.02, 0.50 ± 0.04, and 0.36 ± 0.02 µM, respectively. J78 also produced a shift to the right of the concentration-response curve of U46619 [GenBank] , indicating an antagonistic effect on the TXA₂ receptor. J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation. In addition, J78 potently suppressed TXA₂ formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner but had no effect on the production of PGD₂, indicating an inhibitory effect on TXA₂ synthase. This was supported by a TXA₂ synthase activity assay that J78 concentration-dependently inhibited TXB₂ formation converted from PGH₂. Furthermore, J78 was also able to inhibit the [Ca²⁺]_i mobilization induced by collagen or thrombin at such a concentration that completely inhibited platelet aggregation. Taken together, these results suggest that the antiplatelet activity of J78 may be mediated by TXA₂ receptor blockade with TXA₂ synthase inhibition and suppression of cytosolic Ca²⁺ mobilization.

The cytosolic Ca²⁺ mobilization plays a crucial role in platelet activation and aggregation. During platelet activation, the increase of [Ca²⁺]_i as a result of either Ca²⁺ influx or release from intracellular stores is fundamental to the platelet response to various agonists (Jackson et al., 2003). Accordingly, agents with inhibition of the cytosolic Ca²⁺ mobilization in platelets may suppress the platelet aggregation (Kim et al., 1999; Shah et al., 1999; Kang et al., 2001).

The compounds with backbone of 1,4-naphthoquinone chemical structure have shown a wide variety of pharmacological effects such as antiviral, antifungal, anticancer, and antiplatelet activities (Chen et al., 2002; Lien et al., 2002). In our previous study, we have reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, displayed a potent antithrombotic effect in mice in vivo and antiplatelet activity in vitro as well as in rat ex vivo but had no effect on coagulation system. Available results suggest that antithrombotic effect of J78 may be due to anti-platelet activity (Jin et al., 2004). In the present study, we examined possible antiplatelet mechanism of J78 by measurements of the arachidonic acid liberation and formations of TXB₂, prostaglandin (PG) D₂, and 12-hydroxyl-eicosatetraenoic acid (HETE) from exogenous arachidonic acid in platelets. In addition, the antagonistic effect of J78 on TXA₂-mediated platelet aggregation as well as possible inhibitory effects on TXA₂ synthase activity and cytosolic Ca²⁺ mobilization were also investigated.

	Materials and Methods

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Chemicals. J78 was synthesized as previously described (Jin et al., 2004). Briefly, a solution of 1,4-naphthoquinone (0.01 mol) and 2-bromo-4-fluorophenylamine (0.011 mol) in 150 ml of 95% EtOH was refluxed for 5 h. After the reaction mixture was kept overnight at 4°C, precipitate was collected by filtration. Crystallization of the precipitate from MeOH afforded J78. J78 had color: bright-red crystal, m.p.: 135–137.0°C, IR (KBr, cm^–1): 3315, 1494, 1240, 733; 1H NMR (DMSO-d6): 6.88 to 7.41 (3H, m, benzene ring), 7.49 to 7.93 (3H, m), 11.48 (1H, s, OH), 9.27 (1H, s, NH), MS (m/z): 397 (M+), 288. 1,4-Naphthoquinone and 2-bromo-4-fluorophenyl amine, bovine serum albumin (BSA), collagen, DMSO, Fura-2 AM, and U73122 [GenBank] [1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione] were purchased from Aldrich Chemical Co. (Milwaukee, WI). Thrombin and arachidonic acid were purchased from Chrono-Log Co. (Havertown, PA). TXB₂, PGD₂, 12-HETE, and U46619 [GenBank] were purchased from Cayman Chemical (Ann Arbor, MI). [³H]Arachidonic acid (250 µCi/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The other chemicals were of analytical grade.

Animals. New Zealand white rabbits were purchased from Sam-Tako Animal Co. (Osan, Korea) and acclimatized for 1 week at 24°C and 55% humidity, with free access to a commercial pellet diet obtained from Samyang Co. (Wonju, Korea) and drinking water before experiments. The animal studies have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Chungbuk National University, Korea.

Washed Platelet Preparation. Blood was withdrawn from the ear aorta of male New Zealand white rabbits and collected directly into 0.15 (v/v) of anticoagulant citrate dextrose solution that contained 0.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v). Washed platelet was prepared as previously described (Son et al., 2004). Briefly, platelet-rich plasma (PRP) was obtained by centrifugation of rabbit blood at 230g for 10 min. Platelets were sedimented by centrifugation of the PRP at 800g for 15 min and washed with Hepes buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 5.6 mM glucose, and 3.8 mM Hepes, pH 6.5) containing 0.35% BSA and 0.4 mM EGTA. The washed platelets were resuspended in Hepes buffer (pH 7.4) and adjusted to 4 x 10⁸ cells/ml.

Measurement of Platelet Aggregation in Vitro. Platelet aggregation was measured by using an aggregometer (Chrono-Log Co.) according to the turbidimetry method of Born and Cross (1963). Briefly, washed platelet suspension of rabbits was incubated at 37°C for 4 min in the aggregometer with stirring at 1000 rpm before aggregation was challenged by the addition of collagen (10 µg/ml), thrombin (0.05 U/ml), arachidonic acid (100 µM), and U46619 [GenBank] (1 µM), respectively. The resulting aggregation, measured as the change in light transmission, was recorded for 10 min. To investigate the antagonism of J78 on U46619 [GenBank] -induced rabbit platelet aggregation, concentration-response relationships were determined in the absence or presence of a range of concentrations of J78; for these experiments, indomethacin-treated washed rabbit platelets (50 µM for 3 min) were used to prevent any possible contribution of endogenous arachidonic acid metabolites to platelet aggregation. The extent of inhibition of platelet aggregation is expressed as percentage of inhibition (X) using the following equation: X (percent) = (1 – B/A) x 100%, where A is the maximum aggregation rate of vehicle-treated platelets, and B is the maximum aggregation of sample-treated platelets.

Measurement of TXB₂, PGD₂, and 12-HETE Generation. The TXB₂, PGD₂, and 12-HETE generations were measured as previously described (Son et al., 2004). Briefly, washed platelets (4 x 10⁸ cells/ml) were preincubated with various concentrations of J78 at 37°C for 3 min, and then further incubated with a mixture of [³H]arachidonic acid and unlabeled arachidonic acid (2 µM, 1 µCi/ml) for 5 min. The reaction was terminated by the addition of stop solution containing 2.6 mM EGTA and 130 µM BW755C 3-amino-1-(m-(trifluoromethyl)-phenyl)-2-pyrazoline], a COX and lipoxygenase inhibitor. Lipids were extracted and separated by thin-layer chromatography on Silica Gel G plates (Analtech, Newark, DE) with the following development system: ethyl acetate/isooctane/acetic acid/H₂O (9:5:2:10, v/v/v/v). The area corresponding to each lipid was scraped off, and the radioactivity was determined by liquid scintillation counting (model LS 3801; Beckman Coulter, Fullerton, CA).

TXA₂ Synthase Activity Assay. The TXA₂ synthase activity was assayed as previously described (Son et al., 2004). Briefly, aliquots of PGH₂, in anhydrous acetone, were pipetted into glass tubes, and then the acetone was evaporated under a gentle stream of nitrogen, and PGH₂ was redissolved immediately in ethanol. Platelet suspensions were incubated with the test compounds at 37°C for 3 min prior to the addition of 5 µM PGH₂. The final concentration of ethanol was 0.1% (v/v). At 5 min after the addition of PGH₂, the incubations were terminated by the addition of cooling EGTA (2 mM) and centrifuged at 12,000g at 4°C for 4 min. The amount of TXB₂ in the supernatants was assayed by a commercial enzyme immunoassay kit according to the manufacturer's instructions (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). TXA₂ synthase activity is reflected by the production of TXB₂.

Measurement of [Ca²⁺]_i. Cytosolic Ca²⁺ measurements employed the fluorescent dye Fura-2, which involved incubating the platelets with cell permeant acetoxymethyl ester. Rabbit platelets (isolated as described above) were incubated with 2 µM Fura-2/AM at room temperature for 1 h (on a rocking platform) in the loading buffer (137 mM NaCl, 27 mM KCl, 0.4 mM NaH₂PO₄, 10 mM HEPES, 12 mM NaHCO₃, 5.5 mM dextrose, and 0.35% BSA, pH 7.4). Excess Fura-2/AM was removed by centrifugation (500g for 10 min), and the platelets were suspended in fresh buffer, without added EGTA. Aliquots of platelet suspension (2.5 ml) were added to 4-ml cuvettes containing a Teflon-coated stirrer bar (Chrono-Log Co.). Just before [Ca²⁺]_i measurements were performed, Ca²⁺ was added back to the buffer to a final concentration of 1 mM, and then samples (various concentrations in 25 µl) and agonists were added. The measurements of [Ca²⁺]_i were performed at room temperature in an MSIII fluorometer (Photon Technology International, Monmouth Junction, NJ) using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. [Ca²⁺]_i was calculated by using the SPEX dM3000 software package.

Measurement of Arachidonic Acid Liberation. The arachidonic acid liberation was measured as previously described (Son et al., 2004). Briefly, PRP was preincubated with [³H]arachidonic acid (1 µCi/ml) at 37°C for 1.5 h and then washed as described above. The [³H]arachidonic acid-prelabeled platelets (4 x 10⁸ cells/ml) were pretreated with 100 µM BW755C, various concentrations of J78, and U73122 [GenBank] (50 µM) at 37°C for 3 min in the presence of 1 mM CaCl₂, and then stimulated with collagen (50 µg/ml). The reaction was terminated by the addition of chloroform/methanol/HCl (200:200:1, v/v/v). Lipids were extracted and separated by thin-layer chromatography on silica gel G plates with the following development system: petroleum ether/diethyl ether/acetic acid (40/40/1, v/v/v). The area corresponding to each lipid was scraped off, and the radioactivity was determined by liquid scintillation counting.

Statistical Analysis. The experimental results were expressed as mean ± S.E.M. A one-way analysis of variance was used for multiple comparison (Sigma Stat; SPSS Inc., Chicago, IL). If there was a significant variation between treated groups, Dunnett's test was applied. Differences with P < 0.05 were considered statistically significant.

	Results

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Effect of J78 on Rabbit Platelet Aggregation in Vitro. As shown in Fig. 1, J78 concentration-dependently inhibited collagen (10 µg/ml)-, U46619 [GenBank] (1 µM)-, thrombin (0.05 U/ml)-, and arachidonic acid (100 µM)-challenged washed rabbit platelet aggregation, with IC₅₀ values of 0.32 ± 0.01, 0.36 ± 0.02, 0.44 ± 0.02, and 0.50 ± 0.04 µM, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effect of J78 on washed rabbit platelet aggregation. Washed rabbit platelet suspension was incubated at 37°C in an aggregometer with stirring at 1000 rpm, and then J78 was added. After 3 min of preincubation, the platelet aggregation was induced by the addition of thrombin (0.05 U/ml), arachidonic acid (100 µM), collagen (10 µg/ml), or U46619 [GenBank] (1 µM), respectively. The aggregation percentages were expressed as percentage of maximum aggregation induced by respective inducers. Data are expressed as mean ± S.E.M. (n = 4).

Effects of J78 on Conversions of Arachidonic Acid to TXB₂, PGD₂, and 12-HETE in Rabbit Platelet. As shown in Fig. 2, J78 concentration-dependently suppressed TXB₂ generation, which reflected the formation of TXA₂, induced by the addition of [³H]arachidonic acid in intact rabbit platelets. The TXB₂ formations were inhibited by 21.8, 35.4, 53.7, and 76.3% at the concentrations of 0.2, 0.4, 0.6, and 0.8 µM, respectively. J78, however, has no effect on PGD₂ generation. These results suggest that J78 may selectively inhibit activity of TXA₂ synthase rather than that of COX because TXA₂ and PGD₂ are simultaneously produced from arachidonic acid through the COX pathway. In addition, J78 has no effect on 12-HETE production (data not shown), suggesting that lipoxygenase pathway was not involved in the antiplatelet effect of J78.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Effects of J78 on conversions of arachidonic acid to TXB2 and PGD2. Washed rabbit platelets were preincubated with various concentrations of J78 for 3 min without CaCl2 and then further incubated with a mixture of [3H]arachidonic acid and the unlabeled arachidonic acid (2 µM) for 5 min. The [3H]thromboxane B2 generation was measured as described under Materials and Methods. Data are expressed as mean ± S.E.M. (n = 3). *, P < 0.01; **, P < 0.005 versus corresponding stimulus control.

Effect of J78 on TXA₂ Synthase Activity in Rabbit Platelet. The conversion of arachidonic acid to TXA₂ in platelets requires the action of two enzymes, COX and TXA₂ synthase. TXA₂ synthase catalyzes the conversion of PGH₂ to TXA₂ in platelets. By utilizing PGH₂, it is possible to circumvent the COX step during arachidonic acid metabolism. The addition of increasing concentrations of PGH₂ to washed rabbit platelet suspensions produced a concentration-dependent increase of TXB₂ (data not shown). Thus, washed rabbit platelet suspensions containing PGH₂ are adequate for the direct evaluation of the TXA₂ synthase inhibitor. In washed rabbit platelet suspensions, the level of TXB₂ in unstimulated platelets was about 15 ng/4 x 10⁸ platelets. After incubation of washed platelet suspensions with PGH₂ (5 µM) at 37°C for 5 min, TXB₂ formation was increased to 88.8 ng/4 x 10⁸ platelets. As shown in Fig. 3, J78 inhibited the conversion of PGH₂ into TXB₂ by 9.8, 20.3, 45.3, and 63.1% at concentrations of 0.2, 0.4, 0.6, and 0.8 µM in washed rabbit platelet suspensions, respectively. Imidazole, a typical TXA₂ synthase inhibitor, also markedly inhibited the conversion of PGH₂ into TXB₂.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of J78 on TXA2 synthase activity. After preincubation of indomethacin (50 µM) at 37°C for 2 min, platelet suspension containing DMSO (0.1%), J78, or imidazole (50 mM) was further incubated for 3 min, and then 5 µM PGH2 was added. At 5 min after the addition of PGH2, the incubations were terminated by the addition of cooling EGTA (2 mM) and centrifugation at 12,000g at 4°C for 4 min. TXB2 formation in the supernatants was determined by enzyme immunoassay. TXA2 synthase activity is reflected by the production of TXB2, which is presented as mean ± S.E.M. (n = 3). *, P < 0.05; **, P < 0.01 versus corresponding stimulus control.

Effect of J78 on U46619 [GenBank] -Induced Rabbit Platelet Aggregation. When J78 was preincubated with washed rabbit platelets for 3 min, it reduced platelet aggregation elicited by various concentrations of U46619 [GenBank] under COX blockade with indomethacin in a concentration-dependent manner. At concentrations of 0.36 and 0.80 µM, J78 produced a shift to the right of the concentration-response curve of U46619 [GenBank] , suggesting an antagonism on the TXA₂ receptor (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effect of J78 on rabbit platelet aggregation induced by U46619 [GenBank] . Indomethacin (50 µM)-treated rabbit platelets for 3 min were used to prevent any possible contribution of endogenous arachidonic acid metabolites to platelet aggregation. Washed rabbit platelets were incubated with J78 or DMSO (0.1%) at 37°C for 4 min, and then U46619 [GenBank] was added to trigger aggregation. The peak level of aggregation was measured for 4 min after the addition of stimulator. The percentage of aggregation was calculated assuming the maximum value of the control (absence of drug) produced by U46619 [GenBank] (1 µM) to be 100%. Each data point is expressed as mean ± S.E.M. (n = 3).

Effect of J78 on [Ca²⁺]_i in Rabbit Platelet. The representative traces in which two different agonists were added to induce [Ca²⁺]_i mobilization were shown in Fig. 5. The effect of J78 on [Ca²⁺]_i mobilization was observed after 3 min of incubation with platelet before adding the respective inducers. Collagen induced a slow but stable increase of [Ca²⁺]_i, which reached the peak level of 300 µM after 5 min, whereas thrombin caused a rapid but transient increase in [Ca²⁺]_i. Treatment of the platelet suspension with J78 (0.8 µM) almost completely inhibited the elevation of the [Ca²⁺]_i in response to collagen and thrombin, respectively. The right panels in A and B (Fig. 5) are the average of three times separated experiments, similar to that shown in left panels in A and B.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Effects of J78 on [Ca2+]i in collagen- and thrombin-stimulated rabbit platelet. Calcium (1 mM) was added to the platelet suspension 10 s before data collection started (zero time). J78 solution was added to yield a final concentration of 0.8 µM in the platelet suspension. Collagen (10 µg/ml) or thrombin (0.05 U/ml) was added 3 min later. The traces shown are from a representative experiment; similar results were obtained from separate experiments conducted in triplicate, and average data are presented in right panels in A and B. **, P < 0.01 versus corresponding stimulus control.

Effect of J78 on Collagen-Induced Arachidonic Acid Liberation in Rabbit Platelet. As shown in Fig. 6, pretreatment of J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation in [³H]arachidonic acid-prelabeled rabbit platelets by 2.6, 16.8, 33.7, and 55.6% at concentrations of 0.2, 0.4, 0.6, and 0.8 µM, respectively. U73122 [GenBank] , a phospholipase C inhibitor that was used as a positive control, completely blocked arachidonic acid liberation at a concentration of 50 µM.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of J78 on collagen-induced arachidonic acid liberation in rabbit platelet. [3H]Arachidonic acid-prelabeled platelets were incubated with various concentrations of J78 or U73122 [GenBank] at 37°C for 2 min in the presence of 50 µM BW755C and then stimulated with 50 µg/ml collagen for 2 min. Liberated [3H]arachidonic acid was determined as described under Materials and Methods. Each point is expressed as mean ± S.E.M. (n = 3). *, P < 0.05; **, P < 0.01 versus corresponding stimulus control.

	Discussion

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Platelet aggregation is a complex process, and it is generally considered that platelet activation is mainly mediated through adhesiveness of platelets to the site of injury and through the action of endogenous agonists such as ADP, collagen, and thrombin, followed by the release of TXA₂, which acts as an amplifying factor in the platelet aggregation (Jackson et al., 2003; Farndale et al., 2004). The important role of TXA₂ has been demonstrated by the clinical effectiveness of aspirin in the prevention of cardiovascular diseases such as acute coronary syndromes (Awtry and Loscalzo, 2000; Catella-Lawson et al., 2001; Jneid et al., 2003). In the present study, we demonstrate that the antiplatelet activity of J78, an antithrombotic agent, may be mediated by TXA₂ receptor blockade with TXA₂ synthase inhibition and suppression of [Ca²⁺]_i mobilization.

It is well known that U46619 [GenBank] , which is a TXA₂ mimic, acts directly on the TXA₂ receptor to induce G protein-coupled phospholipase C activation, resulting in an increase of [Ca²⁺]_i and protein kinase C activation (Jackson et al., 2003). Similarly, arachidonic acid, which acts directly on membrane COX enzyme pathway to produce TXA₂, mediates platelet activation in the same way as U46619 [GenBank] (Parise et al., 1984). From the platelet aggregation study (Fig. 1), J78 inhibited TXA₂-mediated platelet aggregation such as arachidonic acid- and U46619 [GenBank] -induced aggregation in a concentration-dependent manner, suggesting that J78 may interfere with the TXA₂ synthesis or its action directly. Therefore, the effect of J78 on the generation of TXA₂ was first determined by using [³H]arachidonic acid in intact rabbit platelet. As shown in Fig. 2, J78 concentration-dependently inhibited [³H]TXA₂ formation, whereas it had no effect on the production of [³H]PGD₂. These results indicate that J78 may selectively inhibit the activity of TXA₂ synthase rather than that of COX because TXA₂ and PGD₂ are simultaneously produced from arachidonic acid through COX pathway in platelets. It was also confirmed by TXA₂ synthase activity assay that J78 potently reduced PGH₂-, a precursor of the PGs and TXA₂, mediated TXB₂ formation (Fig. 3). Although the inhibition of TXA₂ synthesis by TXA₂ synthase inhibition is theoretically rational to inhibit platelet aggregation, considering that accumulated PGH₂, the precursor of TXA₂, can interact with the same receptor (TXA₂/PGH₂ receptor) as TXA₂ to induce platelet activation, it seems that J78 may also have any inhibitory effect on the TXA₂/PGH₂ receptor directly. Therefore, the possible inhibitory effect of J78 on TXA₂/PGH₂ receptor was investigated. Indeed, it completely inhibited platelet aggregation induced by the stable TXA₂ mimic, U46619 [GenBank] , in rabbit platelets by shifting, in a parallel way, the concentration-response curve to U46619 [GenBank] to the right (Fig. 4), suggesting that J78 may specifically block platelet TXA₂/PGH₂ receptor, possibly in a competitive manner. It has also been reported that other drugs possessing the dual property of TXA₂/PGH₂ receptor antagonistic and TXA₂ synthase inhibitory effects act also as competitive antagonists of U46619 [GenBank] -induced aggregation (Chang et al., 1997; Rolin et al., 2001; Miyamoto et al., 2003). These results correlated well with the in vivo antithrombotic effect of J78 on the murine pulmonary thrombosis (Jin et al., 2004), the lethal effect of collagen plus epinephrine on which is caused by massive occlusion of microcirculation of lung by platelet thromboembolism or by vasoconstriction due to the release of TXA₂ and PGF₂ from activated platelets.

In addition, it seems that J78 may also have an inhibitory effect on cytosolic Ca²⁺ mobilization because J78 inhibited calcium ionophore, A23187 [GenBank] -induced platelet aggregation concentration-dependently (Jin et al., 2004). Concomitant with aggregation data, in the platelets loaded with Fura-2 AM, J78 completely blocked the cytosolic Ca²⁺ mobilization induced by collagen and thrombin at a concentration of 0.8 µM, which is sufficient to inhibit platelet aggregation completely (Fig. 5). Thrombin and collagen, both of which are strong agonists, have different platelet aggregation mechanisms (Colman et al., 1994). Thrombin interacts with platelet through a specific receptor belonging to the superfamily of receptors that are coupled to G proteins and phospholipase C, producing diacylglycerol, which stimulates protein kinase C closely linked to secretion. Inositol trisphosphate is also produced and plays a role in increasing [Ca²⁺]_i (Lapetina, 1990), whereas collagen induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and phospholipase C2, which results in [Ca²⁺]_i increase, shape change, and granule release; adhesion is partly and aggregation is largely dependent on ADP and TXA₂/PGH₂ release (Farndale et al., 2004). Considering that inhibition of TXA₂ synthesis was only partly effective to inhibit the collagen- or thrombin-mediated platelet aggregation, and several compounds with the same 1,4-naphthoquinone backbone as J78 have been reported to inhibit the rise of [Ca²⁺]_i by suppression of phosphoinositide breakdown in various cell types including platelet (Chang et al., 1993; Wang and Kuo, 1997; Chen et al., 2002), it is reasonable to speculate that antiplatelet activity of J78 was not only mediated by the TXA₂/PGH₂ receptor antagonism and TXA₂ synthase inhibition but also by the inhibition of cytosolic Ca²⁺ mobilization, possibly by interfering with the phosphoinositide breakdown. This was indirectly supported by the arachidonic acid liberation assay that J78 inhibited collagen-mediated arachidonic acid liberation from [³H]arachidonic acid-prelabeled rabbit platelets in a concentration-dependent manner (Fig. 6). In fact, in lower concentrations (1–20 µg/ml) of collagen-mediated platelet activation, inhibition of phosphoinositide breakdown was able to block the arachidonic acid liberation completely (Balsinde et al., 2002).

Recent studies have demonstrated that injury-induced vascular proliferation and platelet activation are depressed in mice genetically deficient in the TXA₂ receptor or treated with a TXA₂ antagonist (Cheng et al., 2002). Thus, it is conceivable that J78 might be particularly effective for the improvement of atherothrombotic conditions associated with platelet activation. Drugs such as picotamide and ridogrel that act both as TXA₂ synthase inhibitors and as TXA₂/PGH₂ receptor antagonists have been developed. Picotamide may be an effective drug in patients with peripheral occlusive arterial disease of the lower limbs and cerebral infarction (Coto et al., 1989, 1998). Ridogrel seems to have efficacy in patients with peripheral occlusive arterial disease and acute myocardial infarction (Hoet et al., 1990; Neirotti et al., 1994).

In summary, our results demonstrate that antiplatelet activity of J78 may, at least partly, result from a combination of TXA₂ receptor blockade with TXA₂ synthase inhibition and a suppression of [Ca²⁺]_i mobilization.

	Footnotes

 
This work was supported by a Chungbuk National University grant in 2004 and by the program of the Research Center for Bioresource and Health, from the Ministry of Science and Technology and Korea Science and Engineering Foundation.

doi:10.1124/jpet.104.073718.

ABBREVIATIONS: TX, thromboxane; COX, cyclooxygenase; J78, 2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone; PG, prostaglandin; HETE, 12-hydroxyl-eicosatetraenoic acid; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; U73122 [GenBank] , 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U46619 [GenBank] , 9,11-dideoxy-9,11-methanoepoxy-prostaglandin F₂; PRP, platelet-rich plasma; BW755C, 3-amino-1-(m-(trifluoromethyl)-phenyl)-2-pyrazoline.

Address correspondence to: Dr. Yeo-Pyo Yun, College of Pharmacy, Chungbuk National University, 48 Gaesin-Dong, Heungduk-Gu, Cheongju, Chungbuk, 361-763, Korea. E-mail: ypyun{at}chungbuk.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 

Anonymous (1994) Randomized trial of ridogrel, a combined thromboxane A₂ synthase inhibitor and thromboxane A₂/prostaglandin endoperoxide receptor antagonist, versus aspirin as adjunct to thrombolysis in patients with acute myocardial infarction. The Ridogrel Versus Aspirin Patency Trial (RAPT). Circulation 89: 588–595.[Abstract/Free Full Text]

Awtry EH and Loscalzo J (2000) Aspirin. Circulation 101: 1206–1218.[Free Full Text]

Balsinde J, Winstead MV, and Dennis EA (2002) Phospholipase A(2) regulation of arachidonic acid mobilization. FEBS Lett 531: 2–6.[CrossRef][Medline]

Born GV and Cross MJ (1963) The aggregation of blood platelets. J Physiol 168: 178–195.

Catella-Lawson F, Reilly MP, Kapoor SC, Cucchiara AJ, DeMarco S, Tournier B, Vyas SN, and FitzGerald GA (2001) Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. N Engl J Med 345: 1809–1817.[Abstract/Free Full Text]

Chang TS, Kim HM, Lee KS, Khil LY, Mar WC, Ryu CK, and Moon CK (1997) Thromboxane A₂ synthase inhibition and thromboxane A₂ receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets. Biochem Pharmacol 54: 259–268.[CrossRef][Medline]

Chang YS, Kuo SC, Weng SH, Jan SC, Ko FN, and Teng CM (1993) Inhibition of platelet aggregation by shikonin derivatives isolated from Arnebia euchroma. Planta Med 59: 401–404.[Medline]

Chen X, Yang L, Oppenheim JJ, and Howard MZ (2002) Cellular pharmacology studies of shikonin derivatives. Phytother Res 16: 199–209.[CrossRef][Medline]

Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, Lawson JA, and FitzGerald GA (2002) Role of prostacyclin in the cardiovascular response to thromboxane A₂. Science (Wash DC) 296: 539–541.[Abstract/Free Full Text]

Colman R, Cook J, and Niewiarowski S (1994) Hemostasis and Thrombosis, J.B. Lippincott Company, Philadelphia, PA.

Corti R, Farkouh ME, and Badimon JJ (2002) The vulnerable plaque and acute coronary syndromes. Am J Med 113: 668–680.[CrossRef][Medline]

Corti R, Fuster V, and Badimon JJ (2003) Pathogenetic concepts of acute coronary syndromes. J Am Coll Cardiol 41: 7S–14S.[Abstract/Free Full Text]

Coto V, Cocozza M, Oliviero U, Lucariello A, Picano T, Coto F, and Cacciatore L (1989) Clinical efficacy of picotamide in long-term treatment of intermittent claudication. Angiology 40: 880–885.

Coto V, Oliviero U, Cocozza M, and Milani M (1998) Long-term safety and efficacy of picotamide, a dual-action antithromboxane agent, in diabetic patients with carotid atherosclerosis: a 6-year follow-up study. J Int Med Res 26: 200–205.[Medline]

Farndale RW, Sixma JJ, Barnes MJ, and De Groot PG (2004) The role of collagen in thrombosis and hemostasis. J Thromb Haemost 2: 561–573.[CrossRef][Medline]

Fitzgerald DJ, Roy L, Catella F, and FitzGerald GA (1986) Platelet activation in unstable coronary disease. N Engl J Med 315: 983–989.[Abstract]

Hoet B, Arnout J, Van Geet C, Deckmyn H, Verhaeghe R, and Vermylen J (1990) Ridogrel, a combined thromboxane synthase inhibitor and receptor blocker, decreases elevated plasma beta-thromboglobulin levels in patients with documented peripheral arterial disease. Thromb Haemost 64: 87–90.[Medline]

Jackson SP, Nesbitt WS, and Kulkarni S (2003) Signaling events underlying thrombus formation. J Thromb Haemost 1: 1602–1612.[CrossRef][Medline]

Jin YR, Ryu CK, Moon CK, Cho MR, and Yun YP (2004) Inhibitory effects of J78, a newly synthesized 1,4-naphthoquinone derivative, on experimental thrombosis and platelet aggregation. Pharmacology 70: 195–200.[CrossRef][Medline]

Jneid H, Bhatt DL, Corti R, Badimon JJ, Fuster V, and Francis GS (2003) Aspirin and clopidogrel in acute coronary syndromes: therapeutic insights from the CURE study. Arch Intern Med 163: 1145–1153.[Abstract/Free Full Text]

Kang WS, Chung KH, Chung JH, Lee JY, Park JB, Zhang YH, Yoo HS, and Yun YP (2001) Antiplatelet activity of green tea catechins is mediated by inhibition of cytosolic calcium increase. J Cardiovasc Pharmacol 38: 875–884.[CrossRef][Medline]

Kim HS, Zhang YH, and Yun YP (1999) Effects of tetrandrine and fangchinoline on experimental thrombosis in mice and human platelet aggregation. Planta Med 65: 135–138.[CrossRef][Medline]

Lapetina EG (1990) The signal transduction induced by thrombin in human platelets. FEBS Lett 268: 400–404.[CrossRef][Medline]

Lien JC, Huang LJ, Teng CM, Wang JP, and Kuo SC (2002) Synthesis of 2-alkoxy 1,4-naphthoquinone derivatives as antiplatelet, antiinflammatory and antiallergic agents. Chem Pharm Bull (Tokyo) 50: 672–674.[Medline]

Miyamoto M, Yamada N, Ikezawa S, Ohno M, Otake A, Umemura K, and Matsushita T (2003) Effects of TRA-418, a novel TP-receptor antagonist and IP-receptor agonist, on human platelet activation and aggregation. Br J Pharmacol 140: 889–894.[CrossRef][Medline]

Neirotti M, Molaschi M, Ponzetto M, Macchione C, Poli L, Bonino F, and Fabris F (1994) Hemodynamic, hemorheologic and hemocoagulative changes after treatment with picotamide in patients affected by peripheral arterial disease (PAD) of the lower limbs. Angiology 45: 137–141.

Parise LV, Venton DL, and Le Breton GC (1984) Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A₂/prostaglandin H₂ receptor interaction. J Pharmacol Exp Ther 228: 240–244.[Abstract/Free Full Text]

Rolin S, Dogne JM, Michaux C, Delarge J, and Masereel B (2001) Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles. Prostag Leukotr Ess 65: 67–72.

Shah BH, Nawaz Z, Pertani SA, Roomi A, Mahmood H, Saeed SA, and Gilani AH (1999) Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca²⁺ signaling. Biochem Pharmacol 58: 1167–1172.[CrossRef][Medline]

Son DJ, Cho MR, Jin YR, Kim SY, Park YH, Lee SH, Akiba S, Sato T, and Yun YP (2004) Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway. Prostag Leukotr Ess 71: 25–31.[CrossRef]

Wang JP and Kuo SC (1997) Impairment of phosphatidylinositol signaling in acetylshikonin-treated neutrophils. Biochem Pharmacol 53: 1173–1177.[CrossRef][Medline]

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