Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies

Abstract

The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5'-monophosphate) by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P < 0.05). Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1 μ m(2) (P < 0.001). After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P < 0.001). Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study) and thrombosis formation in vivo (murine model) were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids) to processed foods.

Figures

Figure 1

Bioactive compounds indentified in aqueous fraction from S. lycopersicum by HPLC.

Figure 2

Effects of total extract on platelet aggregation. (a) PRP was incubated with saline or total extract (1 mg/mL) as indicated for 3 min, prior to measuring human platelet aggregation induced by ADP 8 µM, collagen 1.5 μg/mL, AA (arachidonic acid 1 mM) or TRAP-6 30 µM, and (b) PRP was incubated with saline or total extract (1 mg/mL) for 20, 60, and 180 seconds, prior to measuring human platelet aggregation induced by ADP 8 µM. The graph depicts the mean ± SEM of n = 3 experiments. *P < 0.05. PRP: platelet-rich plasma.

Figure 3

Aqueous fraction inhibited platelet activation. Platelet activation was measured by percentage of annexin V binding (flow cytometry). PRP was incubated with saline or aqueous fraction (1 mg/mL) for 10 min, prior to measuring human platelet activation induced by agonist (collagen/ADP). The graph depicts the mean ± SEM of n = 3 experiments. *P < 0.05. PRP: platelet-rich plasma.

Figure 4

Effect of aqueous fraction on spreading of human platelets on collagen-coated surfaces. In this experiment, washed platelets were employed. Platelet area (µm2) mean ± SEM was acquired from 4 consecutive fields using a Carl Zeiss LSM 700 confocal microscope. **P < 0.01 and ***P < 0.001.

Figure 5

Aqueous fraction inhibited platelet aggregation and ATP secretion. (a) saline and (b) aqueous fraction (1 mg/mL). Luciferin/luciferase reagent and then ADP 8 µM were added to PRP to induce aggregation and secretion, which were recorded in real time using the lumi-aggregometer. Each aggregation curve is representative of multiple traces obtained from three separate platelet donors. The graph depicts the mean ± SEM of n = 3 experiments. **P < 0.01. PRP: platelet-rich plasma.

Figure 6

Effect of aqueous fraction on collagen-induced platelet adhesion and aggregation under arterial flow conditions. Citrate-anticoagulated blood was preincubated with saline or aqueous fraction (1 mg/mL) for 1 hour and then was perfused over plaque-coated surfaces for 10 min at room temperature at a shear rate of 1000 s−1. (a) Time lapse of 10 min at 1000 s−1, at 30 sec intervals, representative flow images at 40x, (b) the intensity (CTCF) over a time lapse, and (c) bar diagram (values are mean ± SEM; n = 3). ***P < 0.001.

Figure 7

Effect of SQ22536 on platelet aggregation induced by ADP. PRP suspension was incubated with ADP or aqueous fraction plus ADP or pretreated with SQ22536 for 3 min, followed by addition of aqueous fraction and ADP. The graph depicts the mean ± SEM of n = 3 experiments. ***P < 0.001. PRP: platelet-rich plasma.

Figure 8

Effect of aqueous fraction on release of sCD40L from platelets. In this experiment, washed platelets were employed. The graph depicts the mean ± SEM of n = 3 experiments. ***P < 0.001.

Figure 9

Aqueous fraction inhibited arterial thrombosis formation. (a) Representative images of thrombus formation after laser-injured vascular injury in mouse mesenteric artery, saline control group (n = 5), ASA (acetylsalicylic acid) (200 mg/Kg, n = 5), and aqueous fraction (200 mg/Kg, n = 5) to 60 min and (b) representative time course changes of thrombus growth rate.

Figure 10

Administration of aqueous fraction inhibiting ADP-induced platelet aggregation in healthy subjects. Platelet aggregation in PRP induced by ADP 4 µM was studied before (basal) and four hours after oral administration of aqueous fraction (70 mg/kg). The graph depicts the mean ± SEM of n = 6 (each in triplicate) healthy volunteers. *P < 0.05. PRP: platelet-rich plasma.