INTRODUCTION
Platelet activation plays an important role in the hemostasis and arterial thrombosis initiation. Once blood vessels become damaged, subendothelial matrix such as collagen, von Willebrand factor, and thrombin will be exposed or generated at the site of injury, triggering immediate platelet activation and platelet plug formation. Followed by activation of coagulation cascade, the fibrin-containing thrombi rich in aggregated platelets will occlude the site of injury.1 Collagen, which supports the adhesion of platelets to the site of injury via glycoprotein VI (GPVI) and integrin α2β1, induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and phospholipase (PL) Cγ2, which results in cytosolic calcium increase, shape change, and granule release; adhesion is partly dependent, and aggregation is largely dependent, on ADP and prostaglandin (PG) H2/thromboxane (TX) A2 release,2 which recruit more and more platelets to the developing thrombus.1
An increase of free cytosolic calcium level is the major intracellular stimulus involved in platelet aggregation.3,4 The plasma membrane calcium ATPase pump is the main agent of cytosolic calcium removal from platelets that maintains resting [Ca2+]i.5 Increases in [Ca2+]i can lead to liberation of arachidonic acid (AA) from membrane phospholipids and, subsequently, to be catalyzed by cyclooxygenase (COX) and TXA2 synthase to produce TXA2, which amplifies platelet aggregation.6 On the other hand, PGD2, which is synthesized from AA simultaneously with other PGs and TXA2, can inhibit platelet activation by increasing cAMP level as PGI2.7
Epidemiological evidence suggests that tea consumption is associated with a reduced risk of cardiovascular disease.8,9 Epigallocatechin gallate (EGCG), a major component of the green tea catechins (GTC), is the most extensively studied tea polyphenol.10,11 Previous studies have indicated that consumption of green tea extract (1.5-4.5 g, one to three cups) by healthy humans is associated with the appearance of EGCG, ECG, and EC in the plasma, and that the plasma concentration of EGCG is about 4 μM.12-15 It has been reported that EGCG has anticarcinogenic, antimutagenic, antioxidative, antiinflammatory, and antiviral effects.16-19 We have reported previously that GTC and EGCG displayed a potent protective effect on collagen plus epinephrine-induced pulmonary thrombosis in mice by the inhibition of platelet aggregation.20 Although EGCG was reported to inhibit thrombin-mediated platelet activation by hindering the thrombin proteolytic activity,21 the precise antiplatelet mechanism of EGCG is not yet fully understood. In the present study, to further investigate the antithrombotic and antiplatelet activities of EGCG, we undertook to determine the effects of EGCG on FeCl3-induced arterial thrombus formation, platelet aggregation, AA cascade, cytosolic calcium mobilization, and PLCγ2 and protein tyrosine phosphorylation. In addition, we compared the effects of EGCG with those of epigallocatechin (EGC), a structural analogue of EGCG lacking a galloyl group in the 3′ position (Fig. 1), on arterial thrombus formation, platelet aggregation, and intracellular signal transductions.
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FIGURE 1: Chemical structures of (−)-epigallocatechin gallate (EGCG) and (−)-epigallocatechin (EGC).
MATERIALS AND METHODS
Materials
Collagen and arachidonic acid were purchased from Chrono-Log Co. (Havertown, PA). Anti-phospho-PLCγ2 and anti-PLCγ2 polyclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-phospho-tyrosine (4G10) monoclonal antibody was purchased from Upstate (Lake Placid, NY). U73122 was purchased from Tocris (Bristol, UK). TXB2 enzyme immunoassay (EIA) kit and antirabbit-horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Amersham Pharmacia (Buckinghamshire, UK). U46619, TXB2, PGD2, and PGH2 were from Cayman Chemical Co. (Ann Arbor, MI). EGCG, EGC, indomethacin, imipramine, fura-2/AM, vitamin E, and thapsigargin were from Sigma Chemical Co. (St. Louis, MO). [3H]AA (250 μCi/mmol) was from New England Nuclear (Waltham, MA). Other reagents were of analytical grade.
Animals
Male Sprague-Dawley rats (n = 12) were purchased from Dae-Han Biolink Co. (Eum sung, Korea) and acclimated for 1 week at a temperature of 24 ± 1°C and humidity of 55 ± 5%. Male New Zealand white rabbits (n = 8) were purchased from Samtako Bio Korea Inc. (Gyunggi, Korea) and acclimated for at least 1 week at a temperature of 24 ± 1°C and a humidity of 55 ± 5%, with free access to a commercial pellet diet obtained from Samyang Co. (Kangwon, Korea) and drinking water before experiments. Animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Chungbuk National University, Korea.
Washed Rabbit Platelets Preparation and Platelet Aggregation In Vitro Assay
The preparation of washed rabbit platelets was performed as previously described.22 In brief, rabbit blood was withdrawn from the ear artery vessel and collected directly into 0.15 (v/v) of anticoagulant citrate dextrose solution containing 0.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v). Platelet-rich plasma (PRP) was prepared by centrifugation at 230g for 10 minutes at room temperature. Platelets were sedimented by centrifugation of PRP at 2100g for 10 minutes and then washed twice with HEPES buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM glucose, 0.35% bovine serum albumin, and 3.8 mM HEPES, pH 6.5) containing 0.4 mM EGTA. After centrifugation, the pellets were resuspended in HEPES buffer (pH 7.4). The platelet concentration was counted using a Coulter Counter (Coulter Electronics, Hialeah, FL) and adjusted to 3 × 108 platelets per milliliter. Platelet aggregation was measured as previously described.23 Briefly, washed platelet suspensions were incubated at 37°C in the aggregometer, with stirring at 1000 rpm. After incubation with various concentrations of EGCG, EGC (5, 50, 100, and 200 μM), or vitamin E (200 μM) for 5 minutes, platelet aggregation was induced by the addition of collagen (10 μg/mL), AA (100 μM), U46619 (1 μM), or thapsigargin (0.3 μM), respectively. The resulting aggregation measured as the change in light transmission was recorded for 10 minutes.
In Vivo Antithrombotic Activity Assay
Arterial thrombosis model was established as previously described.24 In brief, after being fasted overnight, male Sprague-Dawley rats (250 ± 10 g) were anaesthetized with pentobarbital sodium salt (60 mg/kg, i.p.) and intravenously administrated EGCG or EGC in doses of 6.4 μmoL/kg body weight, which yielded a final blood concentration of 80 μM. A segment of the right carotid artery was exposed and dissected, free of the vagus nerve and surrounding tissues, and fitted on the Doppler flow probe (1-mm diameter). Blood flow was measured with a Doppler velocimeter (Crystal Biotech, Northborough, MA). Thrombosis was induced by placing a 2-mm2 Whatman No. 1 filter paper saturated with 50% FeCl3 on the carotid artery near the probe for 10 minutes. The time needed for occlusion to occur was measured, and occlusion time was assigned a value of 60 minutes for vessels that did not occlude within 60 minutes.
Measurement of AA-Mediated TXA2 and PGD2 Formations
The measurement of conversion of AA to TXA2 and PGD2 was performed as previously described.23 Briefly, washed rabbit platelets (3 × 108 platelets per milliliter) were preincubated with various concentrations of EGCG or EGC at 37°C for 5 minutes and then further incubated with a mixture of [3H]AA (1 μCi/mL) and unlabeled AA (2 μM) for 5 minutes. The reaction was terminated by addition of stop solution (2.6 mM EGTA, 130 μM BW755C). Lipids were extracted and separated by silica gel G thin-layer chromatography (TLC) on silica gel G plates (Analtech, Newark, DE). The plates were developed in ethyl acetate:isooctane:acetic acid:H2O (9:5:2:10, v/v/v/v). The area corresponding to each lipid was scraped off, and radioactivity was determined by liquid scintillation counting (model LS 3801, Beckman, Fullerton, CA).
Measurement of TXA2 Formation
The formation of TXA2 in platelets was measured by determining TXB2, the stable metabolite of TXA2. After the incubation of washed platelets with EGCG or EGC at 37°C for 5 minutes, collagen was added for 5 minutes, and then the reaction was terminated by addition of 50 μM indomethacin and 5 mM EDTA. The amount of TXB2 in medium was determined by using the TXB2 EIA kit, according to the procedure described by the manufacturer (Amersham Pharmacia, Buckinghamshire, UK).
Measurement of AA and Diacylglycerol Liberations
The AA liberation assay was performed as previously described.22 In brief, PRP was incubated with [3H]AA (1 μCi/mL) at 37°C for 1.5 hour and then washed, as described above. The [3H]AA-labeled platelets (3 × 108 platelets per milliliter) were pretreated with 100 μM BW755C (3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline, a dual COX and lipoxygenase inhibitor) and various concentrations of EGCG or EGC at 37°C for 5 minutes in the presence of 1 mM CaCl2; then, they were stimulated by the addition of collagen (10 μg/mL). The reaction was terminated by the addition of chloroform:methanol:HCl (200:200:1, v/v/v). After the mixture was vortexed, 5 mM EGTA (containing 0.1 M KCl) was added. Then, samples were centrifuged at 2100g at 4°C for 10 minutes, and the separated upper phase was removed and evaporated to dryness under nitrogen. Residues were dissolved in chloroform:methanol (2:1, v/v) and were applied to thin-layer chromatography plates. The plates were developed in petroleum ether:diethyl ether:acetic acid (40:40:1, v/v/v). The area corresponding to arachidonic acid or diacylglycerol was scraped off, and radioactivity was determined by liquid scintillation counting.
Measurement of [Ca2+]i
The measurement of [Ca2+]i was performed as previously described.22 Briefly, fura 2/AM (3 μM) was added to platelet-rich plasma, and the mixture was incubated at 37°C for 30 minutes. After washing, fura 2/AM-loaded platelets were resuspended in HEPES buffer containing 1 mM CaCl2 and adjusted to a concentration of 3 × 108 platelets per milliliter. Platelets were incubated with 200 μM EGCG or EGC for 5 minutes, and then collagen (10 μg/mL) or thapsigargin (0.3 μM) was added. The measurement of [Ca2+]i was performed at room temperature in an MSIII fluorometer (Photon Technology International, Birmingham, NJ) using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. [Ca2+]i was calculated by using the SPEX dM3000 software package (Photon Technology International).
Immunoblotting Assay
Western blotting analysis was performed to establish whether EGCG and EGC affect PLCγ2 and protein tyrosine phosphorylation. Washed platelets were pretreated with EGCG, EGC, or vehicle for 5 minutes, and then collagen or thapsigargin was added for 5 minutes, respectively. The reaction was terminated by the addition of LaemmLi sample buffer, and the mixture was then boiled for 5 minutes and analyzed on a 7.5% SDS-PAGE. Immunoblotting assays were performed as previously described.22 For immunoblotting, proteins were electrically transferred to the polyvinylidene difluoride membrane for 80 minutes at 120 mA. Blots were incubated for 4 hours with 5% (w/v) BSA in TBS to block residual protein-binding sites. Immunodetection of total- and phospho-PLCγ2 and protein tyrosine phosphorylation were detected by using total- and anti-phospho-PLCγ2 (Q-20, 1 μg/mL), and anti-phospho-tyrosine (4G10) antibodies in TBS containing 5% BSA for 4 hours, respectively. The primary antibody was removed, and blots were washed in TBS with 0.05% Tween-20, three times. To detect the primary antibody, blots were incubated with alkaline phosphatase-conjugated anti-rabbit antibody diluted to 1:5000 in TBS containing 5% BSA for 5 hours, and then washed five times in TBS with 0.05% Tween-20. After the blots had been exposed to enhanced chemiluminescence reagents (Amersham, Piscataway, NJ) for 5 minutes, they were then exposed to hyper film-enhanced chemiluminescence for 5 minutes. The intensities of total- and phospho-PLCγ2, and phospho-tyrosine bands were quantified by Scion Image for Windows (Scion Corporation, Frederick, MD).
Serotonin Secretion Assay
Serotonin concentration was determined by the fluorimetric method of Holmsen and Dangelmaier.25 To prevent the reuptake of secreted serotonin from the dense-granule contents, imipramine (5 μM) was pretreated in the washed platelet suspension. Washed platelets were pretreated with EGCG or EGC at 37°C for 5 minutes, and then collagen (10 μg/mL) was added. After 5 minutes, the reaction was stopped by the addition of 5 mM EDTA in ice, and then the supernatant was centrifuged at 12,000g for 2 minutes. The supernatant was mixed with 6 M trichloroacetic acid (TCA) and centrifuged at 12,000g for 2 minutes. A 0.6-mL aliquot of TCA supernatant was mixed with 2.4 mL of the solution (0.5% o-phthalaldehyde in ethanol diluted 1:10 with 8 N HCl), placed in a boiling water bath for 10 minutes, and then cooled in ice. The excess lipids were extracted with chloroform, and flurophore was measured at the wavelength of excitation (360 nm) and emission (475 nm). Serotonin creatinine sulfate was used as a standard solution to calculate the extent of serotonin release.
Statistical Analysis
The experimental results were expressed as means ± SE. A one-way analysis of variance (ANOVA) was used for multiple comparison (GraphPad Prism version 4.00 for Windows, San Diego, CA). If there was a significant variation between treated groups, the Dunnett test was applied. The data were considered significant with a probability less than 0.05.
RESULTS
Effects of EGCG and EGC on Washed Rabbit Platelet Aggregation In Vitro
Washed platelets were pretreated with various concentrations of EGCG or EGC (25-200 μM), and then platelet aggregation was induced by the addition of AA (100 μM), U46619 (1 μM), or collagen (10 μg/mL), respectively. As shown in Figure 2, EGCG concentration-dependently inhibited AA-, U46619- and collagen-induced platelet aggregation, with IC50 values of 85 ± 4, 61 ± 3, and 99 ± 4 μM, respectively. In contrast, EGC weakly inhibited collagen-induced platelet aggregation with an IC50 value of 194 ± 5, whereas it failed to inhibit half of AA- and U46619-induced aggregation at a highest concentration of 200 μM. In case of thapsigargin, a plasma membrane Ca2+-ATPase inhibitor, induced platelet aggregation, EGCG displayed a complete inhibition of platelet aggregation at a highest concentration of 200 μM, whereas EGC was not effective at all. The inhibitory potencies of EGCG and EGC on platelet aggregation were also compared with vitamin E, a well-known free radical scavenger. It can be observed that vitamin E displayed a slight inhibition on platelet aggregation at a concentration of 200 μM (Fig. 2E), whereas EGCG showed a complete inhibition, indicating that even though the antioxidant activity of EGCG may be involved in its antiplatelet activity, it may not be a determinant factor.
FIGURE 2: Effects of EGCG and EGC on washed rabbit platelet aggregation. Washed rabbit platelets were incubated at 37°C in an aggregometer with stirring at 1000 rpm, and then EGCG, EGC, or vitamin E was added. After 5 minutes of preincubation, the platelet aggregation was induced by addition of (A) AA (100 μM), (B) U46619 (1 μM), (C) collagen (10 μg/mL), or (D) thapsigargin (0.3 μM), respectively. (E) The antiplatelet potencies of EGCG, EGC, and vitamin E (a well-known antioxidant) were compared at a concentration of 200 μM. (F) In vivo antithrombotic effects of EGCG and EGC. After intravenous administration of EGCG, EGC, or PBS as a control, the carotid artery was subjected to chemical injury by 50% FeCl3. Blood flow was measured with a Doppler velocimeter. The results are expressed as occlusion time (n = 4). Data are expressed as means ± SE (n = 4). Significant difference between EGCG and EGC at *P < 0.05, **P < 0.01.
In Vivo Antithrombotic Effects of EGCG and EGC
To investigate the relevance of EGCG to pathologic and occlusive thrombus formation in vivo, rat carotid artery thrombosis was induced by FeCl3 after intravenous administration of EGCG, EGC, or PBS as a control. After FeCl3 application, the injured vessels were occluded within 18.4 ± 3.7 minutes in the control group and 20.4 ± 3.4 minutes in the EGC-treated group, respectively. However, the occluded time of EGCG-treated groups were significantly prolonged to 40.1 ± 3.7 minutes (P < 0.05, n = 4) (Fig. 2F).
Effects of EGCG and EGC on Conversion of AA to TXA2 and PGD2
Because TXA2 and PGD2 are simultaneously produced from arachidonic acid through the COX pathway, we further determined [3H]AA-mediated [3H]TXA2 and [3H]PGD2 formations. As shown in Figure 3, EGCG has no effect on TXA2 formation; however, it concentration-dependently increased [3H]PGD2 formation. On the other hand, neither [3H]TXA2 nor [3H]PGD2 formation was affected by EGC. Indomethacin, a COX inhibitor used as a positive control, almost completely inhibited [3H]TXA2 and [3H]PGD2 formations at a concentration of 20 μM.
FIGURE 3: Effects of EGCG and EGC on conversion of AA to TXA2 and PGD2 in rabbit platelets. Washed rabbit platelets were preincubated with EGCG, EGC, or indomethacin for 5 minutes without CaCl2 and were then further incubated with a mixture of [3H]AA and the unlabeled AA compound (2 μM) for 5 minutes. The [3H]TXB2 generation was measured as described in the Materials and Methods section. Data are expressed as means ± SE (n = 3). Significantly different from control at *P < 0.05, **P < 0.01.
Effects of EGCG and EGC on Collagen-Induced TXA2 Formation
Because TXA2 is unstable and is quickly converted to TXB2, a stable metabolite of TXA2, the amounts of TXA2 were determined using a TXB2 EIA kit. As shown in Figure 4A, the TXB2 level in resting platelet suspension was lower than 1.5 ng/3 × 108 cells and increased to 118 ng/4 × 108 cells after stimulation with collagen (10 μg/mL). Pretreatment of EGCG inhibited collagen-induced TXB2 formation in the same pattern by which it suppressed platelet aggregation, but had no effect on AA-induced TXB2 formation (data not shown). In the case of EGC treatment, neither collagen- nor arachidonic acid-induced TXA2 formation was inhibited. Indomethacin, a positive control, completely blocked TXB2 formation at a concentration of 20 μM.
FIGURE 4: Effects of EGCG and EGC on collagen-induced TXA2 formation, arachidonic acid and DAG liberation, and serotonin secretion in rabbit platelets. After preincubation of platelet suspension with DMSO, EGCG, EGC, or U73122 for 5 minutes, 10 μg/mL of collagen was added. The reaction was terminated by addition of 50 μM indomethacin and 5 mM ice-cold EDTA, and then the supernatant was obtained by centrifugation at 12,000g for 2 minutes. (A) TXB2 was determined, using an EIA kit. (B) Arachidonic acid and (C) DAG liberation assays were performed, using [3H]AA-labeled platelets. After platelet suspension was incubated with various concentrations of EGCG, EGC, or U73122 (a PLC inhibitor) at 37°C for 2 minutes in the presence of 50 μM BW755C, 10 μg/mL of collagen was added for 2 minutes. [3H]AA and [3H]DAG were separated and determined as described in the Materials and Methods section. (D) The serotonin concentration was determined by a fluorimetric method, as described in the Materials and Methods section. Data are expressed as means ± SE (n = 4). Significantly different from control at *P < 0.05, **P < 0.01.
Effects of EGCG and EGC on Collagen-Induced AA and Diacylglycerol Liberations
Effects of EGCG and EGC on AA liberation were estimated using [3H]AA-labeled platelets. Pretreatment of EGCG significantly decreased AA release by 19.4, 34.8, and 62.2% at concentrations of 25, 100, and 200 μM, respectively. On the other hand, EGC had no effect on AA release for all concentration ranges (Fig. 4B). In addition, the [3H]DAG released from phospholipids was simultaneously measured along with the liberated [3H]AA. Treatment of EGCG concentration-dependently inhibited collagen-induced diacylglycerol formation, whereas EGC was not effective at all (Fig. 4C). U73122 (50 μM), a positive control, also completely inhibited AA and diacylglycerol liberations.
Effects of EGCG and EGC on Collagen-Induced Serotonin Secretion
The serotonin level in resting platelet suspensions was lower than 0.2 μM/4 × 108 cells and increased to 5 μM/4 × 108 cells after addition of collagen (10 μg/mL). As shown in Figure 4D, pretreatment of EGCG significantly inhibited release of serotonin by 14.4, 38.9, and 67.1% at concentrations of 25, 100, and 200 μM, respectively, whereas EGC was not effective at all.
Effects of EGCG and EGC on Collagen- and Thapsigargin-Stimulated [Ca2+]i
Cytosolic calcium mobilization is very important in collagen-mediated platelet aggregation and is mainly IP3 dependent. Because EGCG concentration-dependently inhibited collagen-stimulated diacylglycerol formation, which is simultaneously produced with IP3 by PLC, it can be hypothesized that EGCG may also have an effect on cytosolic calcium mobilization. As shown in Figure 5A, at a highest concentration of 200 μM, collagen-induced cytosolic calcium mobilization was completely blocked by EGCG, whereas it was weakly inhibited by EGC. On the other hand, in thapsigargin-stimulated washed platelets, EGCG completely blocked cytosolic calcium mobilization at a concentration of 200 μM, whereas EGC was not effective at all. These observations correlated well with the results of platelet aggregation.
FIGURE 5: Effects of EGCG and EGC on [Ca2+]i mobilization in rabbit platelets caused by collagen and thapsigargin. CaCl2 was added, to yield a concentration of 1 mM before fura 2/AM-loaded platelets were preincubated with EGCG or EGC (200 μM) for 5 minutes, and then (A) collagen (10 μg/mL) or (B) thapsigargin (0.3 μM) was added. The traces shown are from a representative experiment; similar results were obtained from separate experiments conducted in triplicate, and average data are represented in the upper panels in A and B. Significantly different from control at *P < 0.05, **P < 0.01.
Effects of EGCG and EGC on Intracellular Proteins Activation
As EGCG concentration-dependently inhibited collagen-stimulated diacylglycerol formation and cytosolic calcium mobilization, available data indicated that PLCγ2 may be a promising target for EGCG on collagen-induced platelet aggregation. Therefore, washed rabbit platelets were stimulated with 10 μg/mL of collagen in the presence or absence of EGCG or EGC, and PLCγ2 phosphorylation was detected. As shown in Figure 6A, pretreatment with EGCG at concentrations of 25, 100, and 200 μM significantly inhibited collagen-induced PLCγ2 phosphorylation with the inhibition percentages of 10.1, 45.4, 60.3, and 87.5%, respectively. EGC, although it could inhibit collagen-induced calcium mobilization by about 50% at the highest concentration of 200 μM, had no effect on collagen-induced PLCγ2 phosphorylation. In addition, the effects of EGCG and EGC on levels of protein tyrosine phosphorylation were examined in platelet whole-cell lysates (Fig. 6B). Consistent with previous reports, collagen induced an increase in the protein tyrosine phosphorylation of a number of platelet proteins in comparison with basal levels. Pretreatment of platelets with EGCG for 5 minutes before stimulation with collagen resulted in a significant inhibition on protein tyrosine phosphorylation. Again, EGC had no effect on collagen-stimulated platelet protein tyrosine phosphorylation. It has been reported that platelet membrane Ca2+-ATPase can be phosphorylated on tyrosine 1176 in a physiologically relevant process, and that tyrosine phosphorylation leads to inhibition of Ca2+ efflux during platelet activation. As shown in Figure 6C, thapsigargin stimulated a number of proteins' tyrosine residue phosphorylation, especially on 140 kDa; treatment of EGCG completely blocked thapsigargin-induced proteins' tyrosine phosphorylation, whereas EGC was not effective at all.
FIGURE 6: Effects of EGCG and EGC on PLCγ2 and protein tyrosine activation in rabbit platelets. Washed platelets in suspension were treated with EGCG or EGC at 37°C for 5 minutes with stirring (1000 rpm) in the presence of 50 μM indomethacin and 5 mM ice-cold EDTA, and then collagen (10 μg/mL) or thapsigargin (0.3 μM) were added for 5 minutes. The reactions were terminated by addition of an equal volume of LaemmLi sample buffer, and protein was analyzed with SDS-PAGE. (A) and (B) Collagen-stimulated PLCγ2 and platelet protein tyrosine phosphorylation. (C) Thapsigargin-induced platelet protein tyrosine phosphorylation. Data are expressed as means ± SE (n = 4). Significantly different from control at *P < 0.05, **P < 0.01.
DISCUSSION
In the present study, we provided evidence that antithrombotic and antiplatelet activities of EGCG may be attributable to its modulation of multiple cellular targets, such as inhibitions of PLCγ2, protein tyrosine phosphorylation and AA liberation, and elevation of cellular PGD2 level, as well as maintaining Ca2+-ATPase activity, which may underlie its beneficial effect on the atherothrombotic diseases. In addition, the antithrombotic and antiplatelet activities of EGCG are enhanced by the presence of a gallate moiety esterified at the 3′ position on the C ring compared with EGC (Fig. 1).
In the platelet aggregation assay, EGCG potently inhibited the AA-, U46619-, and collagen-induced washed platelet aggregation in a concentration-dependent manner (Fig. 2). In contrast, EGC only slightly inhibited platelet aggregation induced by U46619 and collagen, whereas it showed little effect on that induced by AA. In addition, we have observed that gallated catechins, including EGCG, GCG, ECG, and CG, present more potent inhibitory effects on platelet aggregation than do catechins without gallate moiety at the 3′ position on C ring (EGC, GC, EC, and C; data not shown). These results were also in agreement with Zheng et al's26 report that EGCG seems more effective in inhibiting hypertrophy of vascular smooth-muscle cells than does EGC. We have also reported that catechins that have a galloyl group in the 3′ position of the catechin structure, including ECG, CG, and EGCG, are able to suppress both the PDGF-Rβ-mediated signal-transduction pathway and VSMC proliferation, whereas catechin compounds without a galloyl group, such as catechin and EC, have no effect.27 Considering that the antioxidant activity of EGCG contributes a great impact to its biological functions on various oxidative stress-related cellular events, we have compared the effect of EGCG on platelet reactivity with vitamin E, a well-known free radical scavenger. As shown in Figure 2E, vitamin E displayed a weak inhibition on platelet aggregation at a concentration of 200 μM, whereas EGCG showed complete inhibition, indicating that even though the antioxidant activity of EGCG may be involved in its antiplatelet activity, it may not be a determinant factor. The in vivo antithrombotic effect of EGCG was also examined by using FeCl3-mediated rat artery thrombosis, which is composed of fibrin, activated platelets, and entrapped erythrocytes. This is the type of thrombus that is found in the coronary arteries after sudden death and acute myocardial infarction.28,29 In this model, ferric chloride induces an oxidative injury to expose the subendothelial matrix. Platelet interacts with collagen and von Willebrand factor in the matrix via platelet membrane GPIb-V-IX and αIIbβ3. Glycoprotein VI binding to collagen is required for platelet activation, and activated platelets undergo calcium mobilization and the release of ADP and TXA2 to accelerate platelet recruitment and activation and thrombus formation.1 As shown in Figure 2F, administration of EGCG significantly increased the occlusion time, whereas EGC-treatment was not effective at all, which indirectly indicated that EGCG was able to inhibit thrombus formation in vivo via inhibition of platelet aggregation. Thus, available data indicate that the presence of gallate moiety in the 3′ position is very important for the biological effects of green tea catechins.
In platelet activation in response to various stimuli, TXA2 is an important mediator in the release, reaction, and aggregation of platelets.1 As shown in Figure 2A, EGCG inhibited AA-induced platelet aggregation in a concentration-dependent manner, whereas EGC only showed weak inhibition. There is a significant difference between EGCG- and EGC treatment in all concentration ranges. As shown in Figure 3A, neither EGCG nor EGC has any effect on AA-mediated TXA2 formation, indicating that both agents have no effect on COX-TXA2 synthase activity. Interestingly, the production of PGD2 was concentration-dependently increased by EGCG, but not by EGC. It has been reported that PGD2 can inhibit platelet aggregation by increasing intracellular cAMP levels.7,30 Thus, the presence of a gallate moiety esterified at carbon 3 of the C ring may contribute greatly to the potency of EGCG on AA-induced platelet aggregation when compared with EGC. On the other hand, in platelet aggregation induced by U46619-, a TXA2 mimic, EGCG also displayed a more potent inhibition than did EGC (Fig. 2B). It has been reported that flavonoids, such as apigenin, luteolin, and genistein, can inhibit platelet function through binding to TXA2 receptors,31-33 which relies on structural features such as the presence of the double bond in C2-C3 and a keto group in C4.31 In addition, catechin, which lacks the double bond in C2-C3 and a keto group in C4, displays a higher IC50 value of 160 ± 34 μM.31 In the case of EGCG and EGC, although both lack the double bond in C2-C3 and a keto group in C4, there is still a keto group in the esterified gallate moiety of EGCG, which may somewhat compensate the effect of the keto group in C4 and contribute greatly to the inhibition of U46619-induced platelet aggregation.
In collagen-stimulated platelet aggregation, it can be also observed that EGCG was more potent than EGC (Fig. 2C). Collagen receptor (GPVI and integrin α2β1) activation leads to much tyrosine phosphorylation, followed by PLCγ2 activation2 and a sequential increase of intracellular calcium and protein kinase C activation.34,35 As shown in Figure 4A, EGCG concentration-dependently inhibited collagen-stimulated TXA2 production, whereas EGC was not effective at all. Considering that EGCG has no effect on COX-TXA2 synthase activity, it is reasonable to speculate that the inhibition of EGCG on collagen-induced TXA2 formation may be attributable to the inhibition of AA liberation from phospholipids in plasma membrane. In the present AA liberation study, EGCG was revealed to inhibit collagen-stimulated AA liberation (Fig. 4B), DAG formation (Fig. 4C), and serotonin secretion (Fig. 4D) in the same concentration-dependent manner, whereas EGC was not at all effective in all concentration ranges. Because DAG, which activates platelets via protein kinase C, with final activation of integrin αIIbβ3,3 was produced along with IP3 on the breakdown of membrane phospholipids by PLCγ2 after stimulation with collagen,36 it seems that EGCG may have an inhibition of IP3-mediated cytosolic calcium mobilization. Accordingly, at a concentration of 200 μM, EGCG completely blocked collagen-induced cytosolic calcium mobilization, whereas EGC only showed a partial inhibition (Fig. 5A). Thus, these results suggest that PLCγ2 may be a promising target of EGCG on collagen-stimulated platelet aggregation. As shown in Figure 6A, EGCG potently inhibited collagen-induced PLCγ2 phosphorylation in a concentration-dependent manner as it suppressed platelet aggregation, whereas EGC was not at all effective in all concentration ranges. Stimulation of platelets with collagen results in the activation of a tyrosine kinase-dependent signaling pathway.37,38 Consequently, platelet activation with collagen is accompanied by tyrosine phosphorylation of multiple platelet proteins. We also determined the effect of EGCG and EGC on collagen-stimulated protein tyrosine phosphorylation. It can be observed that EGCG significantly inhibited protein tyrosine phosphorylation, especially on the 10-kDa position, which may correspond to the collagen receptor-complex protein, the FcR γ-chain,37 whereas EGC has little effect. The present results correlated well with the observation that EGCG specifically blocks PDGF-BB-stimulated PDGF-Rβ activation whereas EGC has no effect.27,39
As shown in Figure 2D, EGCG was able to block thapsigargin-induced platelet aggregation and cytosolic calcium mobilization completely, whereas EGC was not effective at all. It has been reported that plasma membrane Ca2+-ATPase plays an essential role in maintaining low cytosolic Ca2+ in platelets, and plasma membrane Ca2+-ATPase is phosphorylated on a tyrosine residue 1176 during thapsigargin-stimulated platelet activation, resulting in inhibition of its ATPase activity.5,40,41 For lack of such a direct antibody, we have to examine the protein tyrosine phosphorylation in thapsigargin-stimulated platelets in the presence of EGCG and EGC. As shown in Figure 6C, at a concentration of 200 μM, EGCG reduced thapsigargin-mediated protein tyrosine phosphorylation, including 140-kDa protein tyrosine phosphorylation, which may correspond to the plasma membrane Ca2+-ATPase,5 whereas EGC was not effective at all at this concentration. Our results demonstrate, again, that the absence of a gallate moiety esterified at carbon 3′ on the C ring resulted in the differential effect of EGCG and EGC on thapsigargin-induced protein tyrosine phosphorylation, cytosolic Ca2+ mobilization, and platelet aggregation.
We also compared the effects of garlic acid on collagen- and thapsigargin-stimulated signal transductions. Garlic acid only slightly inhibited collagen-, AA-, U46619-, or thapsigargin-stimulated platelet aggregation at a highest concentration of 200 μM, but it failed to affect such stimuli-induced signal transduction (data not shown), indicating that gallate moiety must be esterified at carbon 3′ on the C ring to exhibit its potentiation on various biological effects.
Taken together, these observations suggest that the antithrombotic and antiplatelet activities of EGCG are mainly attributable to its modulation of multiple cellular targets, such as inhibitions of PLCγ2, protein tyrosine phosphorylation, AA liberation, and elevation of cellular PGD2 levels, as well as maintaining Ca2+-ATPase activity, and that they are enhanced by the presence of a gallate moiety esterified at carbon 3′ on the C ring, which may underlie its beneficial effects on the atherothrombotic diseases.
ACKNOWLEDGMENTS
This work was supported by the Korean Science and Engineering Foundation, and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2005-005-J15002).
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