Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

Abstract

Background and purpose: Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti-platelet activities have shown stability and bioavailability limitations. However, information on the anti-platelet activity of ginsenoside-Rp1 (G-Rp1), a stable derivative of ginsenoside-Rg3, is scarce. We examined the ability of G-Rp1 to modulate agonist-induced platelet activation.

Experimental approach: G-Rp1 in vitro and ex vivo effects on agonist-induced platelet-aggregation, granule-secretion, [Ca(2+) ](i) mobilization, integrin-α(IIb) β(3) activation were examined. Vasodilator-stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G-Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined. KEY RESULT: G-Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G-Rp1 elevated cAMP levels, it dose-dependently suppressed collagen-induced ATP-release, thromboxane secretion, p-selectin expression, [Ca(2+) ](i) mobilization and α(IIb) β(3) activation and attenuated p38(MAPK) and ERK2 activation. Furthermore, G-Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G-Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively.

Conclusion and implications: G-Rp1 inhibits collagen-induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38(-MAPK) inhibition. These data suggest that G-Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation.

Figure 1

Chemical structure of ginsenoside Rp1 (3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosyl dammarane-3β,12β-diol) (C) and its parent compounds ginsenoside Rg3 (A) and dihydroginsenoside-Rg3 (B).

Figure 2

The inhibitory effect of G-Rp1 on thrombin-, collagen- and ADP-induced platelet aggregation. Platelets (3 × 10⁸mL⁻¹) were pre-incubated with or without the G-Rp1 in the presence of 1 mM CaCl₂ for 2 min at 37°C. The platelets were stimulated with collagen (A and B), thrombin (C and D) and ADP (E). The aggregation reaction was terminated at 5 min, and the % aggregation rate was determined. Tracings (A and C) are representative of platelet aggregation at the indicated concentrations, and the bar graphs (B, D and E) are a summary of 6 to 9 independent experiments. Each column shows the mean ± SEM of at least six independent experiments performed. *: P < 0.05, **: P < 0.01 or ***: P < 0.001 versus agonist-activated control.

Figure 3

Effects of G-Rp1 on collagen-activated platelet ATP release, p-selectin expression TXA₂ production and [Ca²⁺]_i concentration. Washed platelets were pre-incubated with G-Rp1 (at the concentrations indicated) and stirred in an aggregometer for 2 min prior to collagen or thrombin stimulation for 5 min, and then the reactions were terminated followed by granule secretion assay. (A) ATP release in response to agonist stimulation was performed and G-Rp1, dose-dependently, suppressed collagen stimulated platelet ATP release. (B and C) Collagen-induced surface p-selectin expression was analysed. G-Rp1 significantly attenuated collagen-activated platelet surface p-selectin expression. (B), a and b represent untreated basal and collagen-stimulated, and c represents G-Rp1 (20 µM). (C) The bar graph shows a summary of four independent experiments. (D) G-Rp1 was pre-incubated with platelets at 37°C for 3 min followed by the addition of collagen (2.5 µg mL⁻¹). The TXB₂ formation was terminated by addition of 2 mM EDTA and 200 µM indomethacin 5 min after the addition of collagen. (E) Platelets were loaded with Fura-2/AM. The platelets (3 × 10⁸ mL⁻¹) were pre-incubated with or without a G-Rp1 in the presence of 1 mM CaCl₂ for 2 min at 37°C. The platelets were stimulated with thrombin (0.1 U mL⁻¹) for 3 min at 37°C. [Ca²⁺]_i levels were determined. Bar graphs show mean ± SEM of at least three independent experiments performed. *: P < 0.05 or **: P < 0.01 versus agonist-activated control.

Figure 4

G-Rp1 inhibits collagen-induced fibrinogen binding to integrin α_IIbβ₃. The inhibitory effect of G-Rp1 on fibrinogen binding to integrin α_IIbβ₃ in collagen-stimulated platelets was examined by flow cytometric analysis. Washed platelets were pretreated with G-Rp1, and then collagen (2.5 µg mL⁻¹) was added together with Alexa Fluor 488-human fibrinogen (200 µg mL⁻¹), and the sample was incubated at 37°C for 15 min. Bar graph represents summary of inhibitory effect of G-Rp1 on fibrinogen binding. *: P < 0.05, **: P < 0.01 versus control.

Figure 5

G-Rp1 elevates basal levels of cAMP activity. Rat washed platelets were stirred either in the presence of vehicle or G-Rp1 (at the concentrations indicated) in an aggregometer; the reaction was terminated and then cAMP enzyme immunoassays were performed. (A) G-Rp1 significantly increased cAMP accumulation in resting platelets in a dose-dependent manner. (B) IBMX pretreatment slightly potentiated G-Rp1-induced cAMP elevation. Results are a summary of at least three independent experiments performed and results in bar graphs are presented as mean ± SEM *: P < 0.05 or **: P < 0.01 versus control.

Figure 6

Effect of G-Rp1 on VASP and PKAαβγ phosphorylation. (A–C) Washed rat platelets (3 × 10⁸ mL⁻¹) were incubated with vehicle, forskolin, G-Rp1, H-89 or LY297002 (at the concentrations indicated) and were stirred in an aggregometer for 10 min before termination. Then, proteins were extracted, separated, transferred to nitrocellulose and immunoblotted using anti-phospho-VASP^Ser239, VASP^Ser157, PKAαβγ and β-actin antibodies. (A) Concentration-dependent effect of G-Rp1 in VASP^Ser−157 (upper lane) and VASP^Ser−239 (middle lane) activation. (B) Effects of forskolin, G-Rp1 or H-89 pretreatment on phospho-VASP^Ser−157 (upper lane) and total-VASP^Ser−157 (middle lane) phosphorylations. (C) Forskolin and G-Rp1 increased, and H-89 and LY297002 suppressed, the expressions of catalytic PKAαβγ (upper lane) and its substrate phospho-VASP^Ser−157 (middle lane), respectively. Images are representative of three independent experiments. Values in bar graphs are means ± SEM of at least four independent experiments performed in triplicate. *: P < 0.05 or **: P < 0.01 versus control.

Figure 7

G-Rp1 attenuated collagen-activated platelet p38 and ERK2 phosphorylations. (A and B) Washed platelets (3 × 10⁸ mL⁻¹) were stirred in an aggregometer with vehicle, G-Rp1, LY297002, U73122, SB203580 or PD98059 at the concentrations indicated for 3 min prior to stimulation with collagen for 5 min before termination of the reactions. Proteins were extracted, separated by SDS-PAGE transferred to nitrocellulose and immunoblotted with antibodies against ERK and p38 MAPKs. Blots were visualized by ECL, and all immunoblots are representative of three to four similar experiments. (A) G-Rp1 dose-dependently attenuated p38 and ERK2 phosphorylations. (B) Effects of pre-incubation of platelets with G-Rp1 or the above indicated inhibitors on collagen-induced ERK2 and p38 phosphorylations at the doses shown in the figure. Images are representative of three independent experiments. Values in bar graphs are means ± SEM of at least four independent experiments performed in triplicate. *: P < 0.05, **: P < 0.01 or ***: P < 0.001 versus control.

Figure 8

G-Rp1 suppresses collagen-induced PLCγ2 and p85 activations and inhibits the tyrosine phosphorylation of multiple components of the GPVI signalling pathway. Washed platelets in the presence of EGTA (1 mM) were incubated with G-Rp1 or vehicle for 5 min and then stimulated with collagen for 3 min. (A) Lysates from G-Rp1 (5–20 µM) treated platelets were immunoblotted to detect the expression levels of PLCγ2 and PI3K (p85) proteins. (B-D) Platelet lysates were immunoprecipitated by incubating overnight with anti-Fyn, anti-Lyn or anti-Syk (B), anti-LAT or anti-PLCγ2 (C), and anti-PI3K or anti-PLCγ2 (D), respectively, and then incubated with protein A-Sepharose (PAS) for 4 h at 4°C. Precipitated proteins were separated by SDS-PAGE and immunoblotted to detect phosphotyrosine residues. (E) PI3K HTRF assay was performed. Each class I PI3Kα, β and δ isoform protein was incubated in the assay buffer containing 10 µM PIP2 and ATP (200 µM) in the presence of G-Rp1 (10–20 µM) and HTRF signal was determined. Equivalent protein loading was verified by reprobing for Fyn, Lyn, Syk, LAT, PLCγ2 or PI3K. Data are representative of three separate experiments. Images are representative of three independent experiments. Values in bar graphs are means ± SEM of at least four independent experiments performed in triplicate. *: P < 0.05, **: P < 0.01 or ***: P < 0.001 versus control.

Figure 9

The effect of G-Rp1 on thrombus formation in rats and bleeding time in mice. The arteriovenous shunt model was used, and the blood circulation in the cannula shunt was carried out for 15 min, and the thrombus weight was immediately determined. (A) The G-Rp1 (15, 30 or 50 mg kg⁻¹) or vehicle was given orally 1 h before thrombus induction. (B) The G-Rp1 at the dose of (50 mg kg⁻¹) time-dependently inhibited thrombus formation with the highest inhibition after 30 min of the compound treatment. (C) Mice were administered G-Rp1 (50 mg kg⁻¹), aspirin (a positive control, 10 mg kg⁻¹) or vehicle. The mouse tail was transected to induce bleeding, and tail was immersed vertically into the 0.9% saline solution at 37°C, and then the time between the start of transection to bleeding cessation was recorded as the bleeding time. Bar graphs show mean ± SEM of at least six independent experiments performed. *: P < 0.05; **: P < 0.01 versus control.

Figure 10

Ex vivo effect of G-Rp1 administered orally on the collagen- or ADP-induced platelet aggregation and ATP secretion in mice. G-Rp1 (50 or 100 mg kg⁻¹), aspirin (50 mg kg⁻¹) or vehicle was orally administered for 1 week to mice with the last dose 60 min before blood collection. Platelet aggregation (A) and ATP secretion (B) were induced by collagen or ADP at the indicated concentrations. Results are expressed as mean ± SEM (n= 10). *: P < 0.05 versus control.