Inhibitory Effects of Cytosolic Ca ²⁺ Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP ₃RI and Dephosphorylation of ERK in Human Platelets

Abstract

Intracellular Ca ²⁺ ([Ca ²⁺] _i ) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca ²⁺-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155  μM. G-Ro inhibited strongly thrombin-elevated [Ca ²⁺] _i , which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP ₃RI) (Ser ¹⁷⁵⁶) to inhibit [Ca ²⁺] _i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP ₃RI (Ser ¹⁷⁵⁶) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca ²⁺ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP ₃RI (Ser ¹⁷⁵⁶) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca ²⁺] _i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca ²⁺-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.