Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI 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 (IP3RI) (Ser(1756)) to inhibit [Ca(2+)] i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (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 IP3RI (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.

Figure 1

Chemical structure of ginsenoside Ro. Ginsenoside Ro (G-Ro), an oleanane-type saponin, is contained in Panax ginseng Meyer [19, 20] and is composed of oleanolic acid as aglycone and two glucose and one glucuronic acid as sugar component [19].

Figure 2

Effects of G-Ro on thrombin-induced human platelet aggregation. (a) The concentration threshold of thrombin on human platelet aggregation. (b) Effects of G-Ro on thrombin-induced human platelet aggregation. (c) The inhibitory effects of G-Ro on thrombin-induced human platelet aggregation. (d) The IC₅₀ value of G-Ro in thrombin-stimulated human platelet aggregation. Measurement of platelet aggregation was carried out as described in “Section 2.” The rate of inhibition by G-Ro was recorded as the percentage of the thrombin-induced aggregation rate. The IC₅₀ value of G-Ro was calculated according to the 4-parameter log fit method. The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets.

Figure 3

Effects of G-Ro on cytotoxicity. Measurement of cytotoxicity was carried out as described in “Section 2.” For a positive control, 0.1% Triton X-100 was used to treat platelets. The data are expressed as the mean ± standard deviation (n = 4). NS, not significant versus without G-Ro, control.

Figure 4

Effects of G-Ro on thrombin-induced [Ca²⁺]_i mobilization. (a) Effects of G-Ro on [Ca²⁺]_i level in thrombin-induced platelet aggregation. (b) Effects of G-Ro on [Ca²⁺]_i level in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS). (c) Effects of G-Ro on [Ca²⁺]_i level in the presence of G-kinase inhibitor (Rp-8-Br-cGMPS). [Ca²⁺]_i was determined as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets, ^# p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (155 μM), and ^† p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).

Figure 5

Effects of G-Ro on cAMP and cGMP production. (a) Effects of G-Ro on cAMP production in thrombin-induced platelets. (b) Effects of G-Ro on cGMP production in thrombin-induced platelets. cAMP and cGMP were determined as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets.

Figure 6

Effects of G-Ro on inositol 1, 4, 5-trisphosphate receptor type I (IP₃RI) (Ser¹⁷⁵⁶) phosphorylation. (a) Effects of G-Ro on IP₃RI (Ser¹⁷⁵⁶) phosphorylation. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on IP₃RI (Ser¹⁷⁵⁶) phosphorylation in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). Lane 1, thrombin (0.05 U/mL) + G-Ro (300 μM); Lane 2, thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); Lane 3, thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). Western blotting was performed as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets; ^† p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).

Figure 7

Effects of G-Ro on dephosphorylation of ERK. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). Western blotting was performed as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets.

Figure 8

Effects of G-Ro on ATP and serotonin release. (a) Effects of G-Ro on ATP release in thrombin-activated platelets. (b) Effects of G-Ro on serotonin release in thrombin-activated platelets. (c) Effects of G-Ro on ATP release in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (d) Effects of G-Ro on serotonin release in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). Determination of ATP and serotonin release was carried out as described in “Section 2.” The data are expressed as mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets, ^# p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (155 μM), and ^† p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).

Figure 9

Effects of G-Ro on p-selectin expression. (a) The flow cytometry histograms on p-selectin expression. (A) Intact platelets (base); (B) thrombin (0.05 U/mL); (C) thrombin (0.05 U/mL) + G-Ro (50 μM); (D) thrombin (0.05 U/mL) + G-Ro (100 μM); (E) thrombin (0.05 U/mL) + G-Ro (200 μM); (F) thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on thrombin-induced p-selectin expression (%). (c) The flow cytometry histograms on p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (A) Thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); (B) thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). (d) Effects of G-Ro on thrombin-induced p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS) (%). Determination of p-selectin expression was carried out as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ^∗ p < 0.05 versus the thrombin-stimulated human platelets; ^† p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).