Antiplatelet Activity of Epigallocatechin Gallate Is Mediated by the Inhibition of PLCγ2 Phosphorylation, Elevation of PGD2 Production, and Maintaining Calcium-ATPase Activity

Abstract

We have previously reported that green tea catechins displayed a potent antithrombotic effect by inhibition of platelet aggregation. In the present study, the antiplatelet and antithrombotic activities of epigallocatechin gallate (EGCG), the major catechin derived from green tea, were extensively investigated. EGCG inhibited arterial thrombus formation and U46619-, collagen-, and arachidonic acid (AA)-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 61 +/- 3, 85 +/- 4, and 99 +/- 4 microM, respectively. In line with the inhibition of collagen-induced platelet aggregation, EGCG revealed blocking of the collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, and it caused concentration-dependent decreases of cytosolic calcium mobilization, AA liberation, and serotonin secretion. In addition, the platelet aggregation, intracellular Ca2+ mobilization, and protein tyrosine phosphorylation induced by thapsigargin, a Ca2(+)-ATPase pump inhibitor, were completely blocked by EGCG. Contrary to the inhibition of AA-induced platelet aggregation, EGCG failed to inhibit cyclooxygenase and thromboxane (TX) A2 synthase activities, but it concentration-dependently elevated AA-mediated PGD2 formation. In contrast, epigallocatechin (EGC), a structural analogue of EGCG lacking a galloyl group in the 3' position, slightly inhibited collagen-stimulated cytosolic calcium mobilization, but failed to affect other signal transductions as did EGCG in activated platelets and arterial thrombus formation. These results suggest that antiplatelet activity of EGCG may be attributable to its modulation of multiple cellular targets, such as inhibitions of PLCgamma2, protein tyrosine phosphorylation and AA liberation, and elevation of cellular PGD2 levels, as well as maintaining Ca2(+)-ATPase activity, which may underlie its beneficial effect on the atherothrombotic diseases.

An Update on the Health Benefits of Green Tea

“…Many of the studies on platelet aggregation have been carried out using various animal platelets. In addition to showing that green tea catechins were involved in inhibition of platelet aggregation, studies suggested that catechins may affect several cellular targets that are related to platelet activation, including: through the arachadonic acid pathway, inhibition of a cytoplasmic increase in calcium, decreased thrombaxane A 2 (TXA 2 ) production, and inhibition of cyclooxygenase-1 (COX)-1 [90][91][92][93]. A study using human platelets concluded that EGCG was able to inhibit platelet activation by adenosine diphosphate (ADP) stimulation, and suppressed the p38 MAPK phosphorylation of heat shock protein 27 (HSP27), which would inhibit the release of pro-thrombotic contents from platelets [94].…”

Antiplatelet Activity of Obovatol, a Biphenolic Component of Magnolia Obovata, in Rat Arterial Thrombosis and Rabbit Platelet Aggregation

“…The platelet concentration was counted using a Coulter Counter (Coulter Electronics, Hialeah, FL, USA) and adjusted to 4 10 8 platelets per milliliter. Platelet aggregation was measured as previously described 20) . Briefly, washed platelet suspensions were incubated at 37 in the aggregometer with stirring at 1000 rpm.…”

“…Thromboxane B2, prostaglandin D2 and 12-HETE generations were measured as previously described 20) . Briefly, washed rabbit platelets (4 10 8 platelets/mL) were preincubated with various concentrations of obovatol at 37 for 5 min and then further incubated with a mixture of [ 3 H]-AA (1 Ci/mL) and unlabeled AA (2 M) for 5 min.…”

“…PRP was adjusted to a density of 3 10 8 platelets/ml with PPP. Platelet aggregation was measured as previously described 20) using an aggregometer (Chrono-Log Co., Havertown, PA, USA). ADP (2 M) and collagen (10 g/ml) were used as agonists for platelet aggregation.…”

“…Serotonin concentration was determined by the fluorimetric method of Jin et al 20) . Washed platelets were then pretreated with imipramine, serotonin reuptake inhibitor (5 M) and obovatol at 37 for 5 min, and collagen (10 g/mL) or arachidonic acid (100 M) was subsequently added.…”