Epigallocatechin-3-gallate has an anti-platelet effect in a cyclic AMP-dependent manner

Aim: In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 μg/mL)-stimulated platelet aggregation. Methods: Washed platelets (10 ⁸/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37° in the presence of 2 mM exogenous CaCl2 with or without EGCG or other materials, stimulated with collagen (10 μg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca ²⁺ ([Ca ²⁺]i), thromboxane A ₂ (TXA ₂), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. Results: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca ²⁺]i mobilization and TXA ₂ production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser ¹⁵⁷, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser ²³⁹, a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASPSer157 phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser ¹⁵⁷ through A-kinase activation to inhibit [Ca ²⁺]i mobilization and TXA2 production on collagen-induced platelet aggregation. Conclusions: These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregationmediated thrombotic diseases.