Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

doi:10.1016/j.plefa.2003.12.004

Prostaglandins, Leukotrienes and Essential Fatty Acids

Volume 71, Issue 1, July 2004, Pages 25-31

https://doi.org/10.1016/j.plefa.2003.12.004 Get rights and content

Abstract

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC₅₀ values of 61.0±2.5, 105.0±4.9 and 67.0±3.2 μg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9±6.1% and 95.4±2.2% at the doses of 25 and 50 mg/kg, respectively. [³H]AA liberation induced by collagen in [³H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A₂ (TXA₂) and prostaglandin D₂ (PGD₂) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA₂ synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA₂ formation through the inhibition of AA liberation and TXA₂ synthase.

Introduction

Platelet aggregation is a complex phenomenon that probably involves several intracellular biochemical pathways. When activated, platelets change shape, aggregate and release the contents of their intracellular granules [1]. The interactions between platelets and blood vessel walls are important in the development of thrombosis and cardiovascular diseases [2], [3], [4]. When blood vessels are damaged, platelet aggregation occurs rapidly to form haemostatic plugs or arterial thrombi at the sites of vessel injury or in regions where blood flow is disturbed. These thrombi are the source of thromboembolic complications of atherosclerosis, heart attacks, stroke, and peripheral vascular disease [5]. Therefore, the inhibition of platelet function represents a promising approach for the prevention of thrombosis.

Platelets are activated by a number of physiological agonists such as collagen, arachidonic acid (AA), thrombin or platelet activating factor (PAF), and undergo a complex cascade of events that results in shape change, secretion, formation of AA metabolites, and aggregation [6]. Two metabolic pathways of AA liberated by Ca²⁺-dependent PLA₂ exist in platelets. One is the cyclooxygenase (COX) pathway that forms thromboxane A₂ (TXA₂) and prostaglandins (PGs), and the other is the lipoxygenase (LOX) pathway that forms hydroxyeicosatetraenoic acids (HETE) and leukotrienes [7]. AA is a membrane-derived fatty acid that is metabolized by COX to PG endoperoxide intermediates such as PGH₂. In platelets, endoperoxides are further metabolized to TXA₂, which is a potent inducer of platelet aggregation and a vasoconstrictor, by TXA₂ synthase [8]. These mediator levels are increased in several thrombotic disorders [9]. Therefore, agents that inhibit the formation or the action of TXA₂ would be potentially useful in the treatment of thrombotic disorders.

Green tea is the unprocessed dried young leaves of Camellia sinensis, also known as Thea sinensis L., which is widely consumed as a beverage. Green tea constituents, especially catechins, exhibit a wide range of pharmacological effects including anticarcinogenic activity and prevention of cardiovascular diseases [10], [11], [12], [13]. We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation [14]. It was also reported that the antiplatelet activity of GTC could be mediated by inhibition of cytoplasmic Ca²⁺ increase [15]. The effects of GTC on vascular smooth muscle tension and ⁴⁵Ca²⁺ uptake in rat aorta have been previously reported [16]. In addition, we also reported that GTC might be a useful agent for the prevention of cerebral ischemia damage [17], [18].

In the present study, the effect of GTC on AA metabolism was examined in order to elucidate a possible antiplatelet mechanism.

Section snippets

Materials

GTC was prepared as previously described [14]. Collagen and AA were from Chrono-Log Co. (Havertown, PA, USA). U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prostaglandin F_2α), TXB₂, PGD₂ and 12-HETE were from Cayman Chemical Co. (Ann Arbor, MI, USA). Indomethacin and imidazole were from Sigma Chemical Co. (St Louis, MO, USA). TXB₂ enzyme immunoassay kit was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). [³H]AA (100 μCi/mmol) was from Perkin-Elmer Life Sciences Inc.

Effect of GTC on platelet aggregation in vitro

Washed rabbit platelets were preincubated with various concentrations of GTC (10–100 μM), and then were exposed to collagen (1 μg/ml), AA (100 μM) or U46619 (1 μM), a TXA₂/PGH₂ receptor agonist, to examine the inhibitory effect of GTC on rabbit platelet aggregation. As shown in Fig. 1, GTC concentration-dependently inhibited the aggregations induced by collagen, AA and U46619. The IC₅₀ values of GTC on collagen-, AA-, and U46619-induced rabbit platelet aggregation were calculated to be 61.0±2.5,

Discussion

We have previously reported that GTC displayed an antithrombotic effect, which might be due to antiplatelet aggregation rather than anticoagulation [14]. It was also reported that the antiplatelet activity of GTC could be mediated by inhibition of cytoplasmic Ca²⁺ increase [15]. In the present study, the effect of GTC on AA metabolism was examined in order to elucidate a possible antiplatelet mechanism of GTC through AA pathway. The effects of GTC on ATP release, AA liberation, TXB₂, PGD₂ and

Acknowledgments

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ2-PG3-21601-002).

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Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

Abstract

Introduction

Section snippets

Materials

Effect of GTC on platelet aggregation in vitro

Discussion

Acknowledgments

Pharmacol. Ther.

J. Am. Coll. Cardiol.

Mutat. Res.

Thromb. Res.

Brain Res. Bull.

Brain Res.

J. Biol. Chem.

The pathogenesis of atherosclerosis

N. Engl. J. Med.

Hyperactive platelets and complications of coronary artery disease

N. Engl. J. Med.

Role of platelets in thrombosis and hemostasis

Can. J. Physiol. Pharmacol.