Association of Quinone-Induced Platelet Anti-Aggregation with Cytotoxicity

Se-Ryun Kim, Joo-Young Lee, Moo-Yeol Lee, Seung-Min Chung, Ok-Nam Bae and Jin-Ho Chung,1

College of Pharmacy, Seoul National University, Shinrim-dong San 56–1, Seoul 151–742, Korea

Received January 9, 2001; accepted April 3, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various anti-platelet drugs, including quinones, are being investigated as potential treatments for cardiovascular disease because of their ability to prevent excessive platelet aggregation. In the present investigation 3 naphthoquinones (2,3-dimethoxy-1,4-naphthoquinone [DMNQ], menadione, and 1,4-naphthoquinone [4-NQ]) were compared for their abilities to inhibit platelet aggregation, deplete glutathione (GSH) and protein thiols, and cause cytotoxicity. Platelet-rich plasma, isolated from Sprague-Dawley rats, was used for all experiments. The relative potency of the 3 quinones to inhibit platelet aggregation, deplete intracellular GSH and protein thiols, and cause cytotoxicity was 1,4-NQ > menadione >> DMNQ. Experiments using 2 thiol-modifying agents, dithiothreitol (DTT) and 1-chloro-2,4-dintrobenzene (CDNB), confirmed the key roles for GSH in quinone-induced platelet anti-aggregation and for protein thiols in quinone-induced cytotoxicity. Furthermore, the anti-aggregative effects of a group of 12 additional quinone derivatives were positively correlated with their ability to cause platelet cytotoxicity. Quinones that had a weak anti-aggregative effect did not induce cytotoxicity (measured as LDH leakage), whereas quinones that had a potent anti-aggregative effect resulted in significant LDH leakage (84–96%). In one instance, however, p-chloranil demonstrated a potent anti-aggregative effect, but did not induce significant LDH leakage. This can be explained by the inability of p-chloranil to deplete protein thiols, even though intracellular GSH levels decreased rapidly. These results suggest that quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect. If this anti-aggregative effect is subsequently followed by depletion of protein thiols, cytotoxicity results.

Key Words: quinones; platelet anti-aggregation; cytotoxicity; protein thiols; glutathione.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood platelets play an important role in hemostasis, thrombosis, and initiation of various cardiovascular diseases. After exposure to the vessel wall by physical and chemical stress, platelets become rapidly activated and platelet aggregation and adhesion occur (Mustard and Packman, 1979Go; Stormorken, 1984Go). The aggregation of platelets is normally controlled by endogenous anti-aggregating factors such as nitric oxide (NO) and prostacyclin (PGI2). If these controlling factors are disrupted by certain diseases or chemical exposures, excessive platelet aggregation can occur, leading ultimately to cardiovascular disease.

To date, several potential mechanisms for inhibition of platelet aggregation have been suggested. These include inhibition of adenosine receptors and thromboxane synthase (Martinez et al., 1992Go; Vittori et al., 1996Go), increased cGMP (Bassenge and Stewart, 1988Go), increased NO-releasing compounds (Civelli et al., 1994Go), and synthesis of prostacyclin analogues (Katsube et al., 1993Go). Depletion of glutathione (GSH) in platelets has been correlated with inhibition of platelet aggregation by several compounds. These include helaniline (a derivative of sesquiterpene lactone) and 1-chloro-2,4-dinitrobenzene (CDNB), which deplete GSH (Bosia et al., 1985Go; Schroder et al., 1990Go), diamide, which oxidizes GSH (Matsuda et al., 1979Go; Patscheke and Worner, 1978Go), and N-ethylmaleimide (NEM), which alkylates GSH (Hill et al., 1989Go).

Quinones are widely distributed in nature and are used clinically as antitumor drugs, components of multivitamin formulations, and anti-allergic agents (Mandel and Cohn, 1996Go; Monks et al., 1992Go; Smith, 1985Go). Recently, quinones have been shown to be platelet anti-aggregative agents by several different mechanisms. Platelet aggregation was reduced by 2-chloro-3-methyl-1,4-naphthoquinone via inhibition of phosphoinositide breakdown (Ko et al., 1990Go). Alternatively, menadione and vitamin K analogues were suggested to interfere with the mobilization and/or utilization of intracellular Ca2+ (Blackwell et al., 1985Go). In addition, 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione inhibited thromboxane A2 (TXA2) synthase (Chang et al., 1997Go), and 2-methoxy-5-methyl-1,4-benzoquinone inhibited TXA2 receptor binding (Lauer and Anke, 1991Go). Other quinone substances, such as derivatives of benzoquinone (Suzuki et al., 1997Go) and naphthoquinone (Lien et al., 1996Go), also prevent platelet aggregation, but the mechanism(s) of action have not been clarified.

It is well known that quinones rapidly deplete intracellular GSH in various tissues including hepatocytes, kidney cells, and platelets (Brown et al., 1991Go; Seung et al., 1998Go; Stone et al., 1996Go). Likewise, several quinones inhibit platelet aggregation within a short period of time as described above. However, the relationship between inhibition of platelet aggregation and depletion of GSH by quinones has not been investigated. Our laboratory is interested in examining the effects of quinones on GSH depletion as a mechanism of platelet anti-aggregation.

Following rapid depletion of GSH, quinones can subsequently deplete protein thiols. We previously demonstrated that protein thiol depletion was closely related with cytotoxicity (Cho et al., 1997Go). Because both the anti-aggregative effect and the depletion of GSH occur within a few minutes following quinone exposure, whereas depletion of protein thiols and overt cytotoxicity occur at later time points, we postulated that anti-aggregation was correlated to depletion of GSH and that this event precedes protein thiol depletion and cytotoxicity. To further investigate this hypothesis, we examined the relationships among platelet anti-aggregation, depletion of GSH, protein thiol depletion, and cytotoxicity using a large number of quinone substances in platelets.


    MATERALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and chemicals.
The following chemicals and purified enzymes were purchased from Sigma Chemical Co. (St. Louis, MO): menadione, 1-chloro-2,4-dinitro-benzene (CDNB), dithiothreitol (DTT), duroquinone, aloe-emodin, glutathione reduced form (GSH), thrombin, ADP, oxidized glutathione (GSSG), triton X-100, trisodium citrate, pyruvic acid, DMSO, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), NADPH, and NADH. The following chemicals were purchased from Aldrich Chemical Co. (Gillingham, Dorset, U.K.): 1,4-benzoquinone, 1,4-naphthoquinone, and 2,3-dichloro-1,4-naphthoquinone. 2- or 6- (1-Oxopentyl)-5,8-dimethoxy-1,4-naphthoquinone was provided by Dr. Byung-Zun Ahn (Chungnam National University, Korea) and quinonelinedione derivatives were provided by Dr. Chung-Kyu Ryu (Ewha Womans University, Korea). All other chemicals were obtained from standard commercial sources.

Animals.
Female Sprague-Dawley rats (Laboratory Animal Center of Seoul National University, Korea) weighing 200–250 g were used throughout all experiments. Prior to experiments, animals were acclimated for 1 week in the laboratory animal facility maintained at constant temperature and humidity with a 12-h light/dark cycle. Food and water were provided ad libitum.

Preparations of platelets.
All procedures were conducted at room temperature, and the use of glass containers and pipettes was avoided. Blood was collected from the abdominal aorta of ether-anesthetized rats using a syringe containing sodium citrate (3.8%, 9:1), and then centrifuged for 15 min at 150 x g at room temperature. Platelet-rich plasma (PRP) was obtained from the supernatant resulting from this relatively low g-force centrifugation. Platelet-poor plasma (PPP) was obtained from the supernatant of a 20 min, 1500 x g centrifugation of the blood cell residue resulting from the first spin. Throughout all experiments, the platelet number was adjusted to 3 x 108 platelets/ml by diluting PRP with PPP.

Measurement of platelet aggregation.
Platelet aggregation was measured by light transmission, with 100% calibrated as the absorbance of PPP and 0% calibrated as the absorbance of PRP. PRP suspension in a silicon-coated cuvette was stirred at 1200 rpm for 1 min before addition of quinones. Dimethylsulfoxide (DMSO) was used as the vehicle for quinones, such that the final concentration of DMSO in the incubation medium was 0.5%. This concentration was shown to have no effect on either platelet aggregation induced by agonists or platelet lysis. Changes in light transmission were detected by a Lumi-aggregometer (Chrono-log Corp., Havertown, PA).

Intracellular glutathione levels.
Sample preparation was accomplished by incubating PRP with 100 µM quinones. One ml-aliquots of quinone-treated PRP (1 x 109 platelets/ml) were centrifuged at 10,000 x g for 20 s at room temperature to obtain a platelet pellet. The pellets were resuspended with 0.6 ml of 0.125 M perchloric acid containing 0.4 mM EDTA and centrifuged at 10,000 x g for 2 min to obtain the acid soluble fraction (supernatant). The supernatant was mixed with 2 M KOH containing 0.3 M MOPS to remove excess perchloric acid and to adjust the pH to 7.0. A 0.2 ml aliquot of this preparation was then assayed for glutathione. Total glutathione levels were determined by the enzymatic recycling method described by Griffith (1980), with the following modification—a higher activity of glutathione reductase (1.5 kU/ml in assay buffer) was used in order to optimize conditions for assaying platelets.

Lactate dehydrogenase leakage.
Leakage of lactate dehydrogenase (LDH) from platelets was measured as described by Bergmeyer et al. (1965). LDH activity was measured in both the incubation medium and platelets (lysed with 0.3% Triton X-100). LDH leakage was expressed as % of total enzyme activity.

Protein thiol levels.
Protein thiol concentrations were measured using a modification of the colorimetric method of Di Monte et al. (1984). One ml of quinone-treated PRP was spun at 10,000 x g for 20 s, and the supernatant was discarded. The pellet was washed once with 5% perchloric acid and then resuspended in 2.5 ml of Tris-EDTA buffer (0.5 M Tris, 5 mM EDTA, pH 7.6). DTNB (250 µM final concentration) was added and, after 20 min, the absorbance was measured at 412 nm. Protein thiol levels were calculated on the basis of a glutathione calibration curve.

Statistical analysis.
The means and standard errors of means were calculated for all treatment groups. The data were subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test to determine which means were significantly different from each other or control. In all cases, a p-value of < 0.05 was used to determine significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The capacity for 3 naphthoquinone compounds, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), menadione, and 1,4-naphthoquinone (1,4-NQ), to prevent platelet aggregation was investigated. PRP was pretreated with 1 of 3 quinones for 3 min and then thrombin, ADP, or collagen was added to activate platelets. Table 1Go summarizes the relative inhibitory capacity of the 3 quinones. Quinone concentrations that resulted in 50% inhibition of platelet aggregation (IC50) or 100% inhibition of aggregation (IC100) were determined. Concentrations of DMNQ as great as 500 µM failed to inhibit aggregation induced by all 3 agonists. On the other hand, menadione and 1,4-NQ consistently inhibited platelet aggregation, with IC100s being 2 times greater than the IC50s, irrespective of the specific agonist. The relative potency of quinones to inhibit platelet aggregation was 1,4-NQ > menadione >> DMNQ.


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TABLE 1 Inhibition of Platelet Aggregation by 3 Quinone Compounds
 
To determine if inhibition of platelet aggregation by quinones was related to the depletion of glutathione (GSH), total GSH levels in PRP were determined following exposure to 100 µM DMNQ, menadione, or 1,4-NQ (Fig. 1Go). DMNQ did not alter intracellular total GSH levels compared to control. In contrast, menadione treatment resulted in depletion of intracellular GSH by 45% by 3 min and complete depletion by 10 min. 1,4-NQ completely depleted intracellular GSH within 3 min. These data were consistent with quinone potency to inhibit aggregation, as summarized in Table 1Go. To confirm the possible role of GSH in platelet aggregation, the effects of a thiol-donating agent (DTT) and a GSH-depleting agent (CDNB) on the inhibition of thrombin-induced platelet aggregation by menadione and 1,4-NQ were investigated (Fig. 2Go). After PRP was pretreated with CDNB or DTT for the indicated time, PRP was treated with approximately IC50s of menadione (140 µM) or 1,4-NQ (40 µM). DTT or CDNB treatment alone did not affect aggregation in platelets (data not shown). Pretreatment with 1 mM DTT almost completely prevented the inhibition of platelet aggregation by menadione, while pretreatment with 0.2 mM CDNB significantly potentiated the inhibitory effect of menadione on platelet aggregation (Fig. 2AGo). A similar pattern was observed for 1,4-NQ's effects (Fig. 2BGo).



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FIG. 1. Effect of quinones on total GSH levels in platelets. Platelet rich plasma (PRP) suspensions were incubated in the presence of 100 µM quinone for 3 or 10 min. Total GSH levels in platelets were determined as described in Methods section. Values represent mean ± SEM from 3 experiments. *Represent significant differences from control (p < 0.05).

 


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FIG. 2. Modification of anti-aggregative effects of menadione and 1,4-naphthoquinone by DTT and CDNB. PRP suspensions were pretreated with 1 mM DTT for 5 min, or 0.2 mM CDNB for 10 min prior to thrombin addition. (A) 140 µM menadione or (B) 40 µM 1,4-naphthoquinone was added to PRP suspension 3 min before thrombin addition. Changes in light transmission were detected by a Lumi-aggregometer. DTT or CDNB alone did not affect thrombin-induced platelet aggregation. Data are representative tracings of 3 independent experiments.

 
Since previous studies demonstrated that quinones can decrease protein thiol levels following rapid depletion of GSH in platelets (Cho et al., 1997Go), total protein thiol levels were measured following addition of 250 µM DMNQ, menadione, or 1,4-NQ (Fig. 3Go). No effect on protein thiols was observed with DMNQ treatment. However, menadione and 1,4-NQ depleted protein thiols in a time-dependent manner, with 1,4-NQ pretreatment more rapidly depleting protein thiol levels than menadione. The order of the 3 quinones at 250 µM to deplete protein thiols was 1,4-NQ > menadione >> DMNQ.



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FIG. 3. Effect of quinones on protein thiol levels in platelets. PRP suspensions were incubated with 250 µM quinone and total protein thiol levels were determined at the indicated time points. Total protein thiol level at time 0 was 78 ± 6 nmol/mg protein. Values represent mean ± SEM from 3 experiments. *Represent significant differences from control (p < 0.05).

 
To investigate the role of protein thiols in the cytotoxic effects of these quinone compounds on platelets, the ability of DMNQ, menadione, or 1,4-NQ to induce platelet cytotoxicity was assessed at various time points by measuring LDH leakage (Fig. 4Go). At equimolar concentrations of 250 µM, DMNQ did not induce LDH leakage, while menadione and 1,4-NQ induced LDH leakage in a time-dependent manner. 1,4-NQ appeared more effective than menadione, since LDH leakage was 70% of total LDH activity by 30 min and 100% of total by 60 min. The order of the 3 quinines at 250 µM to cause cytotoxicity was also 1,4-NQ > menadione >> DMNQ (Fig. 4Go), a similar observation to that describing effects on platelet aggregation, glutathione depletion, and protein thiols depletion.



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FIG. 4. Effect of quinones on LDH release from platelets. All treatments were the same as described in Fig. 3Go and release of LDH was determined as described in Methods section. Values represent mean ± SEM from 3 experiments. *Represent significant differences from control (p < 0.05).

 
In addition, the ability of DTT and CDNB to modulate quinone-induced cytotoxicity was investigated (Fig. 5Go). PRP was treated with menadione or 1,4-NQ following exposure to CDNB and DTT. Neither CDNB nor DTT alone resulted in LDH leakage (data not shown). Similar to the results shown above with GSH (Fig. 2Go), pretreatment with 1 mM DTT almost completely inhibited menadione- and 1,4-NQ-induced cytotoxicity, while pretreatment with 0.2 mM CDNB greatly potentiated the effect of the 2 quinones (Fig. 5Go).



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FIG. 5. Modification of cytotoxicity of menadione and 1,4-naphthoquinone by DTT and CDNB. PRP suspensions were pretreated with 1 mM DTT for 5 min, or 0.2 mM CDNB for 30 min prior to addition of (A) 250 µM menadione or (B) 200 µM 1,4-naphthoquinone (1,4-NQ). LDH release was determined at the indicated time point. DTT or CDNB alone did not induce any significant cytotoxicity in platelets. Values represent mean ± SEM from 3 experiments. *Represent significant differences from corresponding control (p < 0.05).

 
To determine if the apparent association between anti-aggregative effects and cytotoxicity observed with DMNQ, menadione, or 1,4-NQ was applicable to a broad class of quinones, the same 2 parameters (anti-aggregative effect and cytotoxicity) were measured for a number of additional quinone derivatives (Table 2Go). Quinones demonstrating a weak anti-aggregative effect (i.e., IC50 > 250 µM), such as duroquinone, 2-(1-oxopentyl)-5,8-dimethoxy-1,4-naphthoquinone (2-oxoDMNQ), 6-(2,3,4-trifluorobenzyl)-anilino-5,8-quinolinedione (FAQ 1), 6-(3-fluorobenzyl)-anilino-5,8-quinolinedione (FAQ 2), or anthraquinones, did not induce LDH leakage. Conversely, quinones demonstrating a potent anti-aggregative effect (i.e., IC50 = 7.0 µM~137 µM), such as 1,4-benzoquinone, 2,3-dichloro-1,4-naphthoquinone (DCNQ), 6-(1-oxopentyl)-5,8-dimethoxy-1,4-naphthoquinone (6-oxoDMNQ), 6-(2,4-difluorobenzyl)-anilino-5,8-quinolinedionylchloride (FAQ 4), or menadione, induced significant LDH leakage (84–96%). In addition, the most potent anti-aggregative quinone, 2,3-dichloro-1,4-naphthoquinone (i.e., IC50 = 7.0 µM), also most rapidly induced LDH leakage (69.3 ± 2.5% of the total LDH level within 30 min, data not shown). These results indicate that, in general, the more potent the anti-aggregative effect of a quinone substance, the more cytotoxic that quinone substance will be.


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TABLE 2 Correlation between Anti-Aggregative Effect (IC50) and Cytotoxicity (LDH) by Various Quinones
 
An exception to this general rule, however, was p-chloranil. Among the 15 quinones tested, only p-chloranil demonstrated a potent anti-aggregative effect (IC50 = 7.5 µM) with relatively modest LDH leakage (20% of total activity). Since we observed that protein thiols depletion was associated with quinone cytotoxicity (Figs. 3 and 4GoGo), we postulated that the relatively low cytotoxicity of p-chloranil was associated with a minimal capacity to deplete protein thiols. The effects of p-chloranil on total soluble glutathione levels (Fig. 6AGo) and protein thiol levels (Fig. 6BGo) in platelets were studied. GSH was rapidly depleted by 100 µM p-chloranil, while concentrations as high as 250 µM p-chloranil failed to induce significant depletion of protein thiols with even much longer durations of exposure.



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FIG. 6. Depletion of GSH and protein thiols by p-chloranil. PRP suspensions were incubated in the presence of 100 µM p-chloranil (for total GSH) or 250 µM p-chloranil (for protein thiols). Total GSH and protein thiol levels were determined at the indicated time point, respectively. (A) Total GSH, (circle) Control; (triangle) 100 µM p-chloranil. (B) Protein thiols, (circle) Control; (triangle) 250 µM p-chloranil. Values represent mean ± SEM from 3 experiments. *Represent significant differences from corresponding control (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our current research in platelets can be summarized with the following observations: (1) quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect; the more potent the ability to deplete GSH, the more potent the anti-aggregative effect; (2) quinones that subsequently deplete protein thiols eventually lead to cytotoxicity. These observations lead us to conclude that if GSH depletion by quinones is subsequently followed by depletion of protein thiols, the anti-aggregative effect of the quinones may represent an early event of cytotoxicity.

It has been previously reported that GSH plays an important role in platelet aggregation, and that GSH-depleting chemicals inhibit platelet aggregation significantly (Bosia et al., 1985Go; Heptinstall et al., 1987Go; Schroder et al., 1990Go). However, the association between the depletion of GSH and inhibition of aggregation in platelets has not been reported for quinones. Our data in Table 1Go and Figure 1Go demonstrate that the potency of GSH depletion for 3 quinone model compounds (DMNQ, menadione, and 1,4-NQ) correlates well with their potency to inhibit aggregation. In addition, among all the quinones tested, the compounds that inhibit platelet aggregation with IC50s < 250 µM (Table 2Go) also significantly deplete cellular GSH levels within 10 min (data not shown), suggesting that the anti-aggregative effect of quinones is related to GSH depletion.

To date, several mechanisms have been proposed for inhibition of platelet aggregation by GSH depletion, but the exact mechanism remains unclear. Previous research (Schroder et al., 1990Go) has shown that GSH depletion by some substances other than quinones was associated with inhibition of platelet aggregation and suggested that this possibly caused cytoskeletal protein alterations. Our research has shown that GSH depletion by quinones also caused inhibition of platelet aggregation. It is possible that cytoskeletal alterations may be the key mechanism for our observations. This premise is supported by other studies (Bellomo et al., 1990Go; Thor et al., 1988Go) in isolated hepatocytes that showed that quinones induce cytoskeletal damage mediated by depletion of glutathione and protein thiols, leading to plasma membrane blebbing and ultimately cell death.

The relative potency (IC50s and IC100s) for the 3 model naphthoquinones to inhibit platelet aggregation was 1,4-NQ > menadione >> DMNQ, regardless of which of 3 agonists (collagen, ADP, thrombin) was used (Table 1Go). Both menadione and 1,4-NQ were most effective at inhibiting platelet aggregation induced by collagen, as compared to aggregation induced by the other 2 agonists. There is currently no known explanation for why collagen's action is most easily affected by quinones, but this observation has been reported in several studies using quinones (Chang et al., 1997Go; Lien et al., 1996Go). The effects of DTT and CDNB on the inhibition of platelet aggregation by quinones were obtained when platelet aggregation was induced by thrombin (Fig. 2Go). Similar effects, however, were observed using ADP and collagen agonists (data not shown), suggesting that GSH-depletion by quinones in platelets plays a key role in modulating platelet aggregation induced by any of the 3 agonists.

In general, quinones rapidly deplete GSH and subsequently deplete protein thiols in various tissues such as liver, kidney, and heart (Brown et al., 1991Go; Di Monte et al., 1984Go; Tzeng et al., 1994Go). In platelets, menadione also rapidly depletes GSH within 10 min and subsequently depletes protein thiols for up to 2 h, finally leading to cytotoxicity (Cho et al., 1997Go). In an attempt to confirm a role for GSH and protein thiols in platelet aggregation and cytotoxicity, we pre-exposed cells to CDNB and DTT, 2 thiol modifying agents, to determine their ability to modulate thrombin-induced platelet aggregation and quinone-induced cytotoxicity. DTT was used as a donator of intracellular thiols based upon the report that DTT donates sulfhydryl groups to GSH and protein thiols (Sandy et al., 1988Go). CDNB was used to deplete intracellular thiols. CDNB alone did not affect thrombin-induced platelet aggregation. However, pretreatment with CDNB potentiated the inhibitory effects of 1,4-NQ and menadione on platelet aggregation (Fig. 2Go). The major target of CDNB in platelets appears to be GSH (Bosia et al., 1985Go), but in our experimental system, CDNB may also induce alteration of protein thiol levels, since it subsequently depletes protein thiols by 20% throughout 2-h incubation (data not shown). We have demonstrated that pretreatment with CDNB potentiates quinone-induced cytotoxicity in platelets, whereas DTT completely protects (Fig 5Go), suggesting that protein thiols also play a critical role in platelet cytotoxicity as well.

p-Chloranil, an exceptional quinone among 15 quinones tested, has a potent anti-aggregative effect but it induces a weak cytotoxicity. p-Chloranil was effective at depleting intracellular GSH, but did not significantly deplete protein thiols (Fig. 6Go). This modification of intracellular GSH but not protein thiols by p-chloranil resulted in significant anti-aggregative effect in the absence of significant cytotoxicity. These results suggest that some quinones, such as p-chloranil, which selectively deplete GSH without affecting protein thiol levels, may be potentially effective anti-platelet drugs. However, caution is necessary in interpreting the potential use of quinone compounds as anti-platelet drugs because quinones also deplete intracellular GSH in tissues other than platelets and this depletion has itself been shown to be toxic to cells in these tissues. For example, p-hydroxybenzoate ester-induced cytotoxicity in hepatocytes (Nakagawa and Moldeus, 1998Go), 2-amino-5-chlorophenol-induced toxicity in renal cortical slices (Valentovic et al., 1999Go), and glutamate-induced cytotoxicity in brain cells (Pereira and Oliveira, 1997Go) are primarily dependent upon the depletion of intracellular GSH.

In summary, the depletion of GSH by quinones results in the inhibition of platelet aggregation, and the anti-aggregative effect of the quinones may be an early event in cytotoxicity, as represented by protein thiol depletion. As a consequence, although much research has focused on the possibility that quinone substances could be developed as anti-platelet drug therapy, our results suggest that caution must be applied, since it appears that quinone inhibition of platelet aggregation precedes cytotoxicity.


    ACKNOWLEDGMENTS
 
This work was supported by National Research Laboratory (NRL) Program of the Korean Ministry of Science and Technology and by 2000 BK21 project for Medicine, Dentistry and Pharmacy. We thank Dr. David Thompson for all his help to review this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (822) 885-4157. E-mail: jhc302{at}plaza.snu.ac.kr. Back


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 RESULTS
 DISCUSSION
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