College of Pharmacy, Seoul National University, Shinrim-dong San 561, Seoul 151742, Korea
Received January 9, 2001; accepted April 3, 2001
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ABSTRACT |
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Key Words: quinones; platelet anti-aggregation; cytotoxicity; protein thiols; glutathione.
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INTRODUCTION |
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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., 1992; Vittori et al., 1996
), increased cGMP (Bassenge and Stewart, 1988
), increased NO-releasing compounds (Civelli et al., 1994
), and synthesis of prostacyclin analogues (Katsube et al., 1993
). 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., 1985
; Schroder et al., 1990
), diamide, which oxidizes GSH (Matsuda et al., 1979
; Patscheke and Worner, 1978
), and N-ethylmaleimide (NEM), which alkylates GSH (Hill et al., 1989
).
Quinones are widely distributed in nature and are used clinically as antitumor drugs, components of multivitamin formulations, and anti-allergic agents (Mandel and Cohn, 1996; Monks et al., 1992
; Smith, 1985
). 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., 1990
). Alternatively, menadione and vitamin K analogues were suggested to interfere with the mobilization and/or utilization of intracellular Ca2+ (Blackwell et al., 1985
). In addition, 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione inhibited thromboxane A2 (TXA2) synthase (Chang et al., 1997
), and 2-methoxy-5-methyl-1,4-benzoquinone inhibited TXA2 receptor binding (Lauer and Anke, 1991
). Other quinone substances, such as derivatives of benzoquinone (Suzuki et al., 1997
) and naphthoquinone (Lien et al., 1996
), 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., 1991; Seung et al., 1998
; Stone et al., 1996
). 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., 1997). 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.
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MATERALS AND METHODS |
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Animals.
Female Sprague-Dawley rats (Laboratory Animal Center of Seoul National University, Korea) weighing 200250 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 modificationa 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.
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RESULTS |
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DISCUSSION |
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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., 1985; Heptinstall et al., 1987
; Schroder et al., 1990
). However, the association between the depletion of GSH and inhibition of aggregation in platelets has not been reported for quinones. Our data in Table 1
and Figure 1
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 2
) 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., 1990) 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., 1990
; Thor et al., 1988
) 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 1). 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., 1997
; Lien et al., 1996
). The effects of DTT and CDNB on the inhibition of platelet aggregation by quinones were obtained when platelet aggregation was induced by thrombin (Fig. 2
). 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., 1991; Di Monte et al., 1984
; Tzeng et al., 1994
). 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., 1997
). 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., 1988
). 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. 2
). The major target of CDNB in platelets appears to be GSH (Bosia et al., 1985
), 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 5
), 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. 6). 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, 1998
), 2-amino-5-chlorophenol-induced toxicity in renal cortical slices (Valentovic et al., 1999
), and glutamate-induced cytotoxicity in brain cells (Pereira and Oliveira, 1997
) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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