Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway.

INTERLEUKINS; HETEROCYCLIC compounds; WESTERN immunoblotting; CELL receptors; CELLULAR signal transduction; GENE expression; PLATELET aggregation inhibitors; ENZYME-linked immunosorbent assay; DESCRIPTIVE statistics; ADENOSINE monophosphate; PHOSPHORYLATION

Background. Tetramethylpyrazine (TMP) is an alkaloid in Chinese herbal medicine, which possesses antiplatelet activity. TMP inhibits platelet activation in many ways. The platelet P2Y12 receptor for adenosine 5′ diphosphate (ADP) plays a central role in platelet function, hemostasis, and thrombosis. Here, we investigated the inhibitory effect of TMP on P2Y12 receptor-related platelet function. Methods. The inhibitory potential of TMP was assessed using agonist-induced platelet aggregation, flow cytometric analysis of CD62p expression, PAC-1 activation, and fibrin clot retraction. After the P2Y12 receptor-related signaling pathway was inhibited using the blocker, platelet activation was studied by platelet aggregation, CD62p expression, and PAC-1 activation. The secretion of cyclic adenosine monophosphate (cAMP) was measured using enzyme-linked immunosorbent assay (ELISA), and the expression of signaling pathway protein, phosphorylation of vasodilator-stimulated phosphoprotein, and phosphorylation of Akt were investigated using western blotting. The release of platelet inflammatory mediators was measured using ELISA. Results. TMP had an antiplatelet effect by inhibiting ADP-induced aggregation, P-selectin secretion, and glycoprotein (GP) IIb/IIIa expression and reducing the release of atherosclerotic-related inflammatory mediators (sCD40L and IL-1β). TMP decreased the area of clot retraction, reflecting inhibition of GPIIb/IIIa activation. TMP inhibited adenosine diphosphate-induced platelet activation via increased cAMP production, VASPser157 phosphorylation, and Akt dephosphorylation. Conclusion. TMP selectively inhibits ADP-induced platelet activation via P2Y12 receptor-related signaling pathways. [ABSTRACT FROM AUTHOR]

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