# Antiplatelet Activity of Phenolic Compounds-Fortified Merlot Wine and Pure Phenolic Compounds
## 3 Results
### Activation Markers and Platelet Secretion of Wine Extracts and Phenolic Compounds  
Platelet activation markers of the most active fortified wine sample (F8) and of the phenolic compounds epicatechin, catechoin, quercetin, and caffe acid were evaluated. Gallic acid was not evaluated since no statistical activity was observed compared to the control. A high level of P-selectin in platelets is associated with an increased risk of atherosclerosis and vascular pathologies, so we evaluated the expression of this cell adhesion molecule. The results of the study showed that the F8 wine sample, which was the most active in terms of antiplatelet activity, inhibited p-selectin expression induced by the three agonists, collagen, TRAP-6, and ADP, only at 1 mg/mL, the highest evaluated concentration (Figure 3A-C). However, at lower concentrations, the extract did not show significant inhibition of p-selectin expression compared to the platelet-activated condition. In addition, the F8 wine sample at the highest concentration (1 mg/mL) also inhibited ADP-induced GPIIb/IIIa activation (Figure 3C-E). GPIIb/IIIa is an important marker of platelet activation related to aggregation and blood clot formation. These findings suggest that F8 wine may exert its antiplatelet effect by inhibiting the expression of p-selectin, a marker of platelet activation related to the interaction between platelets and leukocytes during the inflammatory response and clot formation. Figure 3: Effect of lyophilized fortified wine (F8) on the secretion of p-selectin (**A–C**) and activation of GP IIb/IIIa (**D–F**) induced by collagen, TRAP-6, and ADP. Platelet activation and secretion markers were determined by flow cytometry using different lyophilized wine concentrations (0.1, 0.25, 0.5, 0.75, and 1 m/mL). Negative control, phosphate-buffered saline (PBS). Platelets were identified as the CD61\({}^{+}\) population. Statistical analysis was performed by one-way ANOVA (Dunnet’s test). * \(p<\) 0.05, **\(p<\) 0.01, and *** \(p<\) 0.001 vs. vehicle vs. activated control (agonist) (\(n=3\) for duplicate). Furthermore, the F8 wine sample also showed GPIIb/IIIa inhibition, which may have important implications in the modulation of platelet aggregation. These results provide a better understanding of the mechanisms involved in the antiplatelet activity of the F8 wine sample. Additionally, the effect of phenolic compounds on platelet activation markers was evaluated (Figure 4). The results of our study revealed that the phenolic compounds evaluated, including catechchin, quercetin, and caffeic acid, showed the ability to modulate the expression of p-selectin, a cell adhesion molecule associated with the risk of atherosclerosis and vascular diseases. Interestingly, catechin significantly inhibited p-selectin expression when platelets were stimulated with TRAP-6 and collagen at concentrations higher than 0.5 mg/mL. On the other hand, epicatechin did not show a significant effect on p-selectin expression in our study. Regarding quercetin, it was found that it inhibited the expression of p-selectin induced by ADP at a concentration of 1 mg/mL. This indicates that quercetin can regulate the expression of this adhesion molecule in response to ADP stimulation. Caffeic acid also showed an inhibitory effect on ADP-stimulated p-selectin expression, and this effect was concentration-dependent. A significant decrease in the expression of p-selectin was observed up to a concentration of 0.5 mg/mL of caffeic acid. These results suggest that caffeic acid may have an impact on the regulation of p-selectin expression in response to ADP stimulation. Once again, and in agreement with platelet aggregation results shown in Figure 2, gallic acids showed no p-selectin activity, confirming that this phenolic compound does not seem to show any anti-platelet activity. Figure 4: Effect of pure free phenolic compounds on the secretion of p-selectin induced by collagen, TRAP-6, and ADP.