Protective mechanisms of guanosine from Solanum lycopersicum on agonist-induced platelet activation: role of sCD40L

Abstract

In the past 30 years, only three natural products have been sources of new drugs with antiplatelet activity. In this study, we have demonstrated for the first time that guanosine from Solanum lycopersicum possesses antiplatelet (secretion, spreading, adhesion and aggregation) activity in vitro and inhibition of platelet inflammatory mediator of atherosclerosis (sCD40L). According to ADP-induced platelet aggregation inhibiting, the total extract residue was fractionated by liquid chromatography/phase separation, affording an aqueous fraction. This fraction was subjected to repeated permeation over Sephadex LH-20 and semi-preparative TLC. The isolated compound finally obtained was identified as guanosine on the basis of its UV-spectra, HPLC and 1H-NMR data. Guanosine concentration dose-dependently (1 to 4 mmol/L) inhibited platelet secretion and aggregation induced by ADP and collagen. Spread of human platelets on collagen in the presence of guanosine was fully inhibited. After incubation of whole blood with guanosine, the platelet adhesion and aggregation under flow conditions was inhibited concentration dependently (0.2 to 2 mmol/L). At the same concentrations that guanosine inhibits platelet aggregation, levels of sCD40L were significantly decreased. Guanosine is thus likely to exert significant protective effects in thromboembolic-related disorders by inhibiting platelet aggregation.

Figure 1

Scheme of the extraction and bioguided fractionation of S. lycopersicum. (A) Bio-directed isolation of band B-guanosine. (B) Platelet antiaggregant activity of extract and fractions from S. lycopersicum. Platelet aggregation was induced by ADP 8 µmol/L and all samples at 1 mg/mL. Saline (negative control) and PGE₁ (positive control). The graph depicts the average ± SEM of n = 3 experiments. All values are statistically significant vs. saline (* p < 0.05).

Figure 2

Guanosine concentration-dependently inhibited platelet ATP secretion. Effect of guanosine on ADP (8 µmol/L) and collagen (1.5 μg/mL) induced platelet ATP secretion. Saline (negative control) and PGE₁ (positive control). Results were expressed as % inhibition (mean ± SEM, n = 3).

Figure 3

Guanosine concentration-dependently inhibited platelet aggregation. Quantitation of the inhibitory effect of guanosine on platelet aggregation induced by ADP (8 µmol/L) and collagen (1.5 μg/mL). Saline (negative control) and PGE₁ (positive control). Results were expressed as % inhibition (mean ± SEM, n = 3).

Figure 4

Effect of guanosine on spreading of human platelets on collagen-coated surfaces. Platelet area (µm²) mean ± SEM was acquired from 4 consecutive fields using a Carl Zeiss LSM 700 confocal microscope. Control corresponds to saline: maximum spread. * p < 0.05 and ** p < 0.01.

Figure 5

Effect of guanosine on collagen-induced platelet adhesion and aggregation under arterial flow conditions. Citrate-anticoagulated blood was pre-incubated with saline (control), PGE₁ (0.02 mmol/L) or guanosine (0.2 to 2 mmol/L) for 1 hour and then was perfused over plaque-coated surfaces for 10 min at room temperature at a shear rate of 1,000 s⁻¹. (A) timelapse of 10 min at 1,000 s⁻¹, at 30 s intervals, guanosine (2 mmol/L). (B) It shows the intensity (CTCF) over a time lapse. (C) bar diagram (values are mean ± SEM; n = 3). *** p < 0.001.

Figure 6

Effect of guanosine on release of sCD40L from human platelets induced by thrombin. Washed platelets were pretreated with saline, ASA, acetylsalicylic acid 0.3 mmol/L or guanosine (0.4 to 4 mmol/L) for 15 min at 37 °C and then stimulated by thrombin (2 U/mL). The graph depicts the mean ± SEM of n = 3 experiments. *** p < 0.001.

Figure 7

Effect of guanosine on release of sCD40L from platelets induced by haIgG. Washed platelets were pretreated with saline, ASA, acetylsalicylic acid 0.3 mmol/L or guanosine (4 mmol/L) for 15 min at 37 °C and then stimulated by haIgG (500 µg/mL). The graph depicts the mean ± SEM of n = 3 experiments. * p < 0.05, ** p < 0.01.