Antiplatelet Activity of Coumarins: In Vitro Assays on COX-1

Abstract

Atherosclerotic cardiovascular disease is the leading cause of death in developed countries. Therefore, there is an increasing interest in developing new potent and safe antiplatelet agents. Coumarins are a family of polyphenolic compounds with several pharmacological activities, including platelet aggregation inhibition. However, their antiplatelet mechanism of action needs to be further elucidated. The aim of this study is to provide insight into the biochemical mechanisms involved in this activity, as well as to establish a structure-activity relationship for these compounds. With this purpose, the antiplatelet aggregation activities of coumarin, esculetin and esculin were determined in vitro in human whole blood and platelet-rich plasma, to set the potential interference with the arachidonic acid cascade. Here, the platelet COX activity was evaluated from 0.75 mM to 6.5 mM concentration by measuring the levels of metabolites derived from its activity (MDA and TXB₂), together with colorimetric assays performed with the pure recombinant enzyme. Our results evidenced that the coumarin aglycones present the greatest antiplatelet activity at 5 mM and 6.5 mM on aggregometry experiments and inhibiting MDA levels.

Keywords: COX; antiplatelet activity; coumarin; esculetin; esculin; impedance aggregometry; polyphenols.

Figure 1

Chemical structure of the different coumarins assayed: esculin (a), esculetin (b) and coumarin (c).

Figure 2

Graphical representation of the percentage of platelet aggregation for coumarin, esculin and esculetin. Panels (A) (WB samples) and (C) (PRP samples) shows results in AA-induced platelet aggregation. Panels (B) (WB samples) and (D) (PRP samples) shows results in ADP-induced platelet aggregation. Results are expressed as the mean and standard deviation for 10 donors. Error bars represent the standard deviation. * p < 0.05: statistically significant differences in platelet aggregation between samples with and without the tested phenolic compound.

Figure 3

Graphical representation of the percentage of COX activity in AA-induced activated PRP samples after addition of increasing concentrations of indomethacin, as positive control, and assayed coumarins. Results are expressed as the mean and standard deviation for 10 donors. Error bars represent the standard deviation. * p < 0.05: significant differences regarding COX activity with and without the examined substances.

Figure 4

Graphical representation of the percentage of h-COX-1 activity after indomethacin and tested coumarins addition in AA-induced activated samples. Results are expressed as the mean and standard deviation for 10 donors. Error bars represent the standard deviation. * p < 0.05: significant differences on h-COX-1 activity with and without the examined substances.

Figure 5

Graphical representation of the percentage of TXB₂ production at different concentrations of indomethacin and coumarins after CI-induced aggregation. Results are expressed as the mean and standard deviation for 10 donors. Error bars represent the standard deviation. * p < 0.05: significant differences between the basal TXB₂ production with and without the examined substances.

Figure 6

Graphical representation of MDA absorbance in PSS y PRP. Panel (A): in the range from 100 nM to 1000 nM and linear fitting in PRP samples (r = 0.997). Panel (B): in the range from 1 µM to 10 µM and linear fitting in PRP samples (r = 0.994).