Antithrombotic Activities of Green Tea Catechins and (−)-Epigallocatechin Gallate

Thrombosis Research 96 (1999) 229–237

REGULAR ARTICLE

Antithrombotic Activities of Green Tea Catechins and (2)-Epigallocatechin Gallate Won-Seek Kang1, Il-Ho Lim1, Dong-Yeon Yuk1, Kwang-Hoe Chung2, Jong-Bum Park3, Hwan-Soo Yoo1 and Yeo-Pyo Yun1 1 College of Pharmacy, Chungbuk National University, Cheongju, 361-763, Korea; 2Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, 120-752, Korea; 3Sama Pharm. Co., Ltd. Seoul, 135-101, Korea. (Received 5 February 1999 by Editor A. Takada; revised/accepted 27 May 1999)

Abstract The antithrombotic activities and mode of action of green tea catechins (GTC) and (2)-epigallocatechin gallate (EGCG), a major compound of GTC, were investigated. Effects of GTC and EGCG on the murine pulmonary thrombosis in vivo, human platelet aggregation in vitro, and ex vivo, and coagulation parameters were examined. GTC and EGCG prevented death caused by pulmonary thrombosis in mice in vivo in a dose-dependent manner. They significantly prolonged the mouse tail bleeding time of conscious mice. They inhibited adenosine diphosphate- and collageninduced rat platelet aggregation ex vivo in a dosedependent manner. GTC and EGCG inhibited ADP-, collagen-, epinephrine-, and calcium ionophore A23187-induced human platelet aggregation in vitro dose dependently. However, they did not change the coagulation parameters such as activated partial thromboplastin time, prothrombin time, and thrombin time using human citrated Abbreviations: EGCG, (2)-epigallocatechin gallate; GTC, green tea catechins; ADP, adenosine diphosphate; BSA, bovine serum albumin; CMC, carboxylmethyl cellulose; PRP, platelet-rich plasma; PPP, platelet-poor plasma; APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide]; TXA2, thromboxane A2; PGF2a, prostaglandin F2a. Corresponding author: Yeo-Pyo Yun, Ph.D., College of Pharmacy, Chungbuk National University, 48 Gaesin-Dong, Heungduk-Gu, Cheongju, Chungbuk, 361-763, Korea. Tel: 182 (431) 261 2821; Fax: 182 (431) 268 2732; E-mail: ,[email protected]..

plasma. These results suggest that GTC and EGCG have the antithrombotic activities and the modes of antithrombotic action may be due to the antiplatelet activities, but not to anticoagulation activities.  1999 Elsevier Science Ltd. All rights reserved. Key Words: Green tea catechins; (2)-epigallocatechin gallate; Antithrombotic activity; Antiplatelet activity

G

reen tea is the unprocessed dried young leaves of Camellia sinensis, also known as Thea sinensis L. that is widely consumed as a beverage. Green tea, especially green tea catechins, shows pharmacological effects such as anticarcinogenic activity [1–8], antioxidant activity [9,10], anticarcinogenic and related dental activity [11,12], antimicrobial activity [13], and prevention of cardiovascular disease [14–17]. Drinking green tea daily would contribute to maintaining plasma catechin levels sufficient to exert antioxidant activity against oxidative modification of lipoproteins in blood circulation systems [18]. Japanese epidemiologists reported that among patients with greater consumption of green tea, that is, 10 or more cups of green tea per day, a significantly decreased risk of gastric cancer was evident [19]. They also reported that one cup of green tea infusion contained 100–200 mg of polyphenolic compounds and an avid tea drinker in Japan could consume about 1 g of (2)-epigallocatechin gallate (EGCG) per day in green tea. Clinical and experi-

0049-3848/99 $–see front matter  1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00104-8

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mental results showed green tea extract to be a nontoxic and inexpensive agent [20]. It was also reported that green tea inhibited the collagen-induced aggregation of rabbit platelet [21]. The antiplatelet activity of green tea may be useful in the prevention and treatment of vascular diseases, but there is no report showing its antithrombotic activity in vivo or the mechanisms of its action. Platelets play an important role not only in normal hemostasis but also in thrombosis at damaged blood vessels since thrombus formation occurs through the activation and aggregation of platelets [22]. The evidence that arterial thrombi are largely composed of platelet aggregates and the fact that platelets play the major part in both initiation and growth of venous thrombi has led many investigators to postulate that platelet aggregation is major pathogenic mechanism in thrombosis [23–27]. Thus, inhibition of platelet function represents a promising approach for the prevention of thrombosis. Considering the importance of thrombosis in cardiovascular disorders, the search for better antithrombotic strategies continues. We previously reported the effects of green tea catechins (GTC) on vascular smooth muscle tension and 45Ca21 uptake in rat aorta [28]. The authors also reported that EGCG selectively inhibited the platelet-derived growth factor-BB (PDGF-BB)induced intracellular signaling transduction pathway in vascular smooth muscle cells. EGCG also inhibited transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172) [29]. In the present study we examined the antithrombotic activities of GTC and EGCG, a major compound of GTC, and the modes of antithrombotic action.

˚ ) were purchased from Sigma Chemical Co. 90 A (St. Louis, MO, USA). Ethanol, chloroform, ethylacetate, carboxylmethyl cellulose (CMC), and acetonitrile were from Junsei Co. (Tokyo, Japan). Cephalin, thromboplastin, and bovine thrombin were purchased from Instrumentation Laboratory Co. (Milano, Italy).

1.2. Isolation of Catechins from Green Tea Dry green tea leaves (100 g) were extracted with 1 L of 70% ethanol at 90 to 958C for 6 hours. The aqueous extract was then concentrated using vacuum rotary evaporator at 458C until total volume was 100 mL. The concentrate was extracted with H2O twice, and then centrifuged at 23,7003g for 20 minutes. The supernatant was further extracted with chloroform once and ethylacetate three times, successively. The extract was then fractionated by column chromatography using Amber˚ ). The colite XAD-7 (mean pore diameter: 90 A umn was eluted serially with H2O and then 95% ethanol fraction was concentrated using vacuum rotary evaporator at 458C and then lyophilized. The total catechin content of GTC was determined by ultraviolet absorbance method [30,31] and its composition of GTC was determined using highperformance liquid chromatography [32,33].

1.3. Animals

1. Materials and Methods

Male Sprague-Dawley rats and Institute of Cancer Research (ICR) mice were purchased from SamYook Animal Co. (Osan, Korea) and acclimated for 1 week at a temperature of 24618C and a humidity of 5565%. The animals had free access to a commercial pellet diet obtained from Samyang Co. (Wonju, Korea) and drinking water before experiments. Animal experiments were carried out in accordance with the international guideline.

1.1. Materials

1.4. In Vivo Antithrombotic Activity

Green tea (Camellia sinensis) was obtained from Pacific Corporation (Chejudo, Korea). EGCG (purity: minimum 95%), adenosine diphosphate (ADP), epinephrine, collagen, A23187, bovine serum albumin (BSA), MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide], RPMI1640, and Amberite XAD-7 (mean pore diameter:

Antithrombotic activities of GTC and EGCG were investigated by the mouse thromboembolism test according to the method of Di Minno et al. [22]. The method appears well suited for screening potential antithrombotic agents, which act primarily against platelet thromboembolism. When mice were given an i.v. injection of a mixture of collagen

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and epinephrine, they died or remained paralyzed due to the pulmonary thrombi. Briefly male ICR mice weighing about 20–25 g were used after overnight fasting. GTC and EGCG were orally administered at doses of 100, 50, and 10 mg/kg. Collagen (114 mg) plus epinephrine (13.2 mg) solution, which was preestimated about 90% induction of thromboembolism, was injected into the tail vein 90 minutes after oral administration of GTC and EGCG. The other groups were orally administered 50 mg/ kg of aspirin as a positive control, or 0.5% CMC solution as vehicle. The number of dead or paralyzed mice was recorded up to 15 minutes and the percentage of protection was calculated by using the equation [12(dead1paralyzed)/total]3100.

1.5. Tail Bleeding Time in Conscious Mice The bleeding time was measured as described by Hornstra et al. [34]. The bleeding time is designed to determine the ability to form hemostatic plug, in which platelet, plasma factor, and blood vessel wall are involved. In short, 60 minutes after the intraperitoneal injection of GTC (4, 10 mg/kg), EGCG (4, 10 mg/kg), or aspirin (10 mg/kg), the tail of the male ICR mouse (20–25 g) was transected at 2 mm from the tip and 1.5 cm of the distal portion was vertically immersed in saline at 378C.

1.6. In Vitro Antiplatelet Aggregation Activity The blood from healthy volunteers who had not taken any antiplatelet drugs for at least 2 weeks was collected by venopuncture into a plastic flask containing 3.15% sodium citrate (1:9 v/v). Plateletrich plasma (PRP) was obtained by centrifugation of the blood at 1203g for 10 minutes at room temperature. Platelet-poor plasma (PPP) was further centrifuged at 8503g for 15 minutes. The number of platelet in PRP was adjusted to 33108 platelets/mL. The platelet aggregation was measured by turbidimetry using Whole Blood Lumi-Ionized Calcium Aggregometer (Chrono-Log Co., Ltd., Havertown, PA, USA) according to the method of Born and Cross [35]. Human PRP (300 mL) was incubated at 378C for 2 minutes in the aggregometer with stirring at 1,000 rpm and then stimulated with ADP, collagen, epinephrine, and calcium ionophore A23187. Changes in light transmission were recorded for 5 minutes after the addition of the

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aggregating agent. Each inhibition rate was obtained from the maximal aggregation induced by respective agonist at the concentration using the equation: inhibition rate5(12maximal aggregation rate (MAR) of sample-treated PRP/MAR of vehicle-treated PRP)3100. Then the values of IC50 (the 50% inhibition concentration) were calculated from the data using a probit method.

1.7. Ex Vivo Antiplatelet Aggregation Activity Antiplatelet aggregation activity was investigated according to the method of Kimura et al. [36]. Male Sprague-Dawley rats weighing 320–350 g were used after overnight fasting. Rats were orally administered via gastric tube 100 mg/kg of GTC solution or 50 mg/kg of aspirin suspended in 0.5% CMC solution. Blood was collected 90 minutes after sample treatment and PRP was prepared as described above. Platelet aggregation was induced by 32.8 mg/mL of collagen or 1.3 mM of ADP in 300 mL of PRP. Antiplatelet aggregation activity was assayed as described above.

1.8. Coagulation Parameters The plasma clotting times, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured by the modification of the method of Hara et al. [37] using Automated Coagulation Laboratory 100 Instrument (Instrumentation Laboratory Company, Milano, Italy) using PPP obtained by centrifugation of the human citrated blood at 1,2003g for 15 minutes. The plasma was incubated with GTC, EGCG, or heparin for 2 minutes at 378C, respectively. Incubated plasma (100 mL) was mixed with 50 mL of cephaline in the process plate. The coagulation was started with the addition of CaCl2, 100 mL of thromboplastin, and 100 mL of thrombin into the 100 mL of the incubated plasma for APTT, PT, and TT assay, respectively. Anticoagulation activity was evaluated by assaying prolongation of plasma clotting time.

1.9. 3-[4,5-dimethylthiazol-2-yl]-2, 5,diphenyl-tetrazolium bromide (MTT) Assay MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5,-diphenyltetrazolium bromide) assay is based on the intracel-

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lular conversion of a tetrazolium salt into a blue formazan product that can be detected using an enzyme-linked immunosorbent assay plate reader. MTT assay was performed as described by Mosmann [38], with some minor modifications. Briefly, effector and target platelets were incubated together for 0, 30, 60, 90, and 120 minutes at 378C. 10 mL of stock MTT solution was then added to each well, followed by 4-hour incubation (378C, 5% CO2) for MTT reduction. At the termination of the assay, platelets were pelleted by centrifugation and after discarding the supernatant, 100 mL of acidified isopropanol (containing 0.01 N HCl) were added to all wells and mixed thoroughly to dissolve the dark blue crystals. After 30 minutes at room temperature plates were read by a Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using a test wavelength of 570 nm and a reference wavelength of 690 nm. All results were expressed as mean values and standard deviations of at least quadruplicate experiments. Cytotoxicity was determined by the following equation: cytotoxicity (%)5(12sample A 570/control A 570)3100.

1.10. Statistics The difference in the data between treatment and control group was analyzed by the Student’s t-test in the ex vivo antiaggregation activity and x2 test in the in vivo antithrombotic activity, respectively.

2. Results 2.1. Isolation of Catechins from Green Tea The yield of GTC was about 10% and the color of the powder was yellowish-brown. GTC contained at least seven catechins, including (2)-epigallocatechin gallate (25–35%), (2)-gallocatechin (10–20%), (2)-epicatechin gallate (7–8%), (2)-gallocatechin gallate (7–8%), (2)-epigallocatechin (4–5%), (2)-epicatechin (4–5%), (2)-catechin (1–2%), among others.

2.2. In Vivo Antithrombotic Activity

Table 1. Effects of GTC and EGCG on the pulmonary thrombosis in mice Sample Control GTC

EGCG Aspirin

No. killed or paralyzed/ no. tested

Protection (%)

Vehicle 100 50 10 50 10 50

18/21 3/20** 7/20** 9/15* 7/23** 6/11* 10/19*

14.3 85.0 65.0 40.0 69.1 45.5 47.4

The samples were administered orally 90 minutes before tail vein injection of epinephrine (13.2 mg/mouse) plus collagen (114 mg/mouse). x2 test used to examine the difference between vehicle- and sample-treated gorups. * p,0.01, ** p,0.001.

death due to pulmonary thrombosis (Table 1). The protective effect of GTC on pulmonary thrombosis was dose dependent and the percentages of its protection were 40, 65, and 85 at the doses of 10, 50, and 100 mg/kg, respectively. EGCG also protected the pulmonary thrombosis dose dependently, and the percentages of its protection were 45.5 and 69.1 at the dose of 10 and 50 mg/kg, respectively. The protection percentage of aspirin was 47.4 at dose of 50 mg/kg.

2.3. Effects on the Tail Bleeding Time of Mice One of the roles of platelet aggregation is to stop bleeding. Therefore, the effects of GTC and EGCG on the bleeding time were checked using the mouse tail bleeding system. The tail bleeding time of untreated mice was measured to be 63.963.1 seconds (n510). As shown in Table 2, GTC (218.6610 sec-

Table 2. Effects of GTC and EGCG on the mouse tail bleeding time Tail bleeding time (seconds)

Treatment (mg/kg) Control GTC EGCG Aspirin

GTC administered orally into mice 90 minutes before i.v. injection of the combination of epinephrine and collagen showed protection from paralysis or

Dose (mg/kg)

Vehicle 4 10 4 10 10

63.963.1 162.1611.2* 218.6610.1** 142.3612.2* 195.6617.6** 226.1616.6**

The samples were administered intraperitoneally 60 minutes before the test. The values are expressed as means6SD (n510). * p,0.01, ** p,0.001 as compared with the control.

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Table 3. IC50 values of GTC and EGCG on the human platelet aggregations IC50 Aggregating agents ADP (20 mM) Collagen (50 mg/mL) Epinephrine (20 mM) A23187 (10 mM)

GTC (mg/mL)

EGCG (mg/mL)

Aspirin (mM)

0.8660.08 0.6460.07 0.4860.01 0.4560.03

0.7760.03 0.5360.10 0.4260.16 0.4360.02

.2* 0.1660.04 0.0760.001 .2*

IC50 values were calculated from at least three separate experiments. The results were expressed as mean6SD. * Less than 50% inhibition at 2 mM.

onds) and EGCG (195.6617.6 seconds) prolonged the mouse tail bleeding time compare to control (63.963.1 seconds) and were similar to aspirin (226.1616.6 seconds).

cium ionophore A23187 were 0.86, 0.64, 0.48, and 0.45 mg/mL, and those of EGCG were 0.77, 0.53, 0.42, and 0.43 mg/mL, respectively, while aspirin did not inhibit ADP-induced platelet aggregation at 2 mM (Table 3).

2.4. In Vitro Antiplatelet Aggregation Activity 2.5. Ex Vivo Antiplatelet Aggregation Activity GTC and EGCG inhibited ADP- (20 mM), collagen- (200 mg/mL), epinephrine- (10 mM), and calcium ionophore A23187 (10 mM)-induced human platelet aggregation in a dose-dependent manner. IC50 values of GTC in human platelet aggregation induced by ADP, collagen, epinephrine, and cal-

The inhibitory effect of GTC on the rat platelet aggregation ex vivo was shown in Figure 1. GTC administered orally into rats inhibited ADP- or collagen-induced platelet aggregation by 39.3 or 53.3% at dose of 100 mg/kg, respectively.

2.6. Effects on Coagulation Parameters The effects of GTC and EGCG on coagulation time were evaluated by APTT, PT, and TT assay in human PPP. GTC and EGCG did not change APTT, PT, and TT. However, heparin prolonged APTT 54.460.3 seconds at 1 mM, PT 14.160.0 seconds at 1 mM and TT 13.361.1 seconds at 1 mM, respectively (Table 4).

2.7. Platelet Cytotoxicity The effects of GTC and EGCG on platelet cytotoxicity were evaluated by MTT assay using human washed platelet. GTC and EGCG did not show any cytotoxicity at the final concentration of 2.0 mg/mL at 0, 30, 60, 90, and 120 minutes (Table 5).

Fig. 1. Inhibitory effect of oral administration of GTC on platelet aggregation in rats. GTC (100 mg/kg) was orally administered and platelet aggregation was induced by ADP (1.3 mM) and collagen (32.8 mg/mL). *Significantly different from control at p,0.01. Means6SD, n53–5.

3. Discussion The results of the present study indicate that GTC and EGCG prevented death due to pulmonary thrombosis and prolonged the tail bleeding time

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Table 4. Effects of GTC and EGCG on the human plasma coagulation parameters Sample Control GTC (mg/mL)

EGCG (mg/mL)

Heparin (mM)

Vehicle 0.1 0.5 1 0.1 0.5 1 1 10

APTT (seconds)

PT (seconds)

TT (seconds)

35.560.3 37.360.1 39.461.2 45.360.9 36.360.1 36.560.1 35.461.2 54.460.3* NC

14.560.1 14.460.1 14.960.1 16.160.1 14.460.1 14.560.2 14.660.1 14.160.0 32.360.7*

7.860.3 7.260.0 7.260.1 6.360.1 7.260.1 7.460.2 7.260.1 13.361.1** NC

The results were expressed as mean6SD (n55). NC5no coagulation. Significantly different from control; * p,0.05, ** p,0.01.

in mice in vivo and inhibited human platelet aggregation in vitro and in rats ex vivo, whereas they did not change the coagulation parameters such as PT, APTT, and TT. These results show that the antithrombotic activities of GTC and EGCG may be mediated by the inhibition of platelet aggregation, and they may not directly act on the release of thromboplastin and/or thrombin formation. In the present in vitro study, GTC and EGCG inhibited human platelet aggregations induced by ADP, collagen, epinephrine, and calcium ionophore A23187, whereas aspirin (a reference drug) failed to inhibit ADP- and A23187-induced platelet aggregation. These results suggest that the modes of antiplatelet actions of GTC and EGCG may be different from that of aspirin. Our results of the antiplatelet aggregation induced by collagen are in agreement with the results of Sagesaka-Mitane et al. [21]. They reported that the hot water extract of green tea and tea catechins, especially EGCG, inhibited the collagen-induced aggregation of

Table 5. Cytotoxicity of GTC and EGCG on the human washed platelet O.D. (570 nm)

Cytotoxicity (%)

Time (minutes)

Control

GTC

EGCG

GTC

EGCG

0 30 60 90 120

1.360.0 0.960.1 1.060.0 1.160.0 1.360.0

3.960.0 3.160.1 2.660.0 2.160.1 2.460.0

3.460.0 2.360.1 2.060.0 2.060.0 2.160.0

,0 ,0 ,0 ,0 ,0

,0 ,0 ,0 ,0 ,0

The cytotoxicity was assessed using the (MTT) colorimetric assay. The final concentrations of GTC and EGCG were 2.0 mg/mL.

washed rabbit platelets. Aspirin is an antithrombotic drug widely used for prophylaxis or prevention of recurrence of thrombosis [39] and is assessed to be effective in some cases of stroke and ischemia [40,41]. However, aspirin has several clinical disadvantages, including gastrointestinal side effects and hemorrhage [42]. This appears to be ascribed to the fact that the drug produces its antiplatelet effect by inhibiting cyclooxygenase activity, but at the same time it also affects blood vessels and decreases the production of PGI2, a biological substance that inhibits the formation of thrombi in blood vessels [43]. The results of an ex vivo study in rats indicate that GTC and EGCG show the inhibitory effects on platelet aggregation when administered orally. The results of antithrombotic activity show that GTC and EGCG prevent death due to pulmonary thrombosis induced by platelet aggregation in a dose-dependent manner. Our preliminary data showed that the maximal plasma concentration of GTC and EGCG occurred at 90 minutes when they were administered orally, and at 60 minutes when injected intraperitoneally. Our data are in agreement with other results [17,44–46]. The bleeding time was also examined to investigate the effect of GTC and EGCG on the hemostatic system in blood vessel. GTC and EGCG prolonged the mouse tail bleeding time compared to control, and their prolongation effects were similar to that of aspirin. EGCG, a component of GTC, shows a similar antithrombotic activity compared with GTC in the present study. It is not clear why antithrombotic activities of GTC and EGCG are equivalent. Chen et al. reported that EGCG in GTE showed a 3.6-

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fold higher absorption rate constant than pure EGCG when administered orally. Based on the area under the plasma concentration vs. time curve (AUC) and maximum concentration (Cmax) produced by EGCG, GTE seems to deliver EGCG into the bloodstream more effectively than pure EGCG compound. They postulate that the formation of EGCG complex with other components in GTE may enhance the absorption of EGCG [44]. The preventive effect of green tea consumption against cardiovascular disease was investigated in a cross-sectional study of 1371 Japanese men [47]. In a cross-sectional study of 1306 Japanese men, serum total cholesterol levels were found to be inversely related to the consumption of green tea [48]. Green tea extract, especially EGCG, has demonstrated the inhibition of the incidence of stroke and prolonged the life span in a hypertension model [13], the inhibition of smooth muscle cell proliferation at low concentration in vitro [14], the antiatherosclerotic activity in vivo [15], and a hypocholesterolaemic effect in vivo [16]. It is possible that these prevention and therapy effects of cardiovascular disease may be associated with the antithrombotic activities. Calcium ionophore A23187 has been known to elevate calcium concentration in platelet due to the influx of extracellular calcium across the plasma membrane. The increase in the cytoplasmic calcium concentration has been known to be a determinant in the stimulus-response coupling to any platelet agonist. The increased calcium concentration contributes to the generation of endoperoxides and phosphorylation of a number of proteins necessary for platelet aggregation [49]. Therefore, it is possible that the inhibitory effects of GTC and EGCG on platelet aggregation may be due to the blockade of calcium influx. The lethal effect of aggregating agents on mice is caused by massive occulation of the microcirculation of the lung by platelet thromboembolic or by vasoconstriction due to an increase of thromboxane A2 (TXA2) and prostaglandin F2a (PGF2a) in platelets [50–52]. It is noteworthy that modulation of TXA2 and PGF2a concentration in platelet would play a critical role in GTC- and EGCG-induced antiplatelet aggregation. Recently, we found that GTC and EGCG decreased TXA2 and PGF2a in human PRP (data not shown). The mechanism of TXA2/PGF2a decrease by as-

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pirin is due to the inhibition of cyclooxygenase, whereas the mechanism by GTC and EGCG is not clear yet. We postulate that the mechanism of TXA2/PGF2a decrease by GTC and EGCG may be associated with calcium and cAMP from the in vitro data. We are performing further studies to elucidate the antiplatelet mechanism of GTC and EGCG at the cellular level. In conclusion, these results suggest that GTC and EGCG have the antithrombotic activities and the modes of antithrombotic actions may be due to the antiplatelet activities, but not to anticoagulation activities. This work was supported by a grant of the Good Health R&D Project, the Ministry of Health and Welfare, R.O.K.

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