Contribution of polyphenols in wine grape extract to the inhibition of serine protease activity and proteinase-activated receptor 1-mediated Ca2+ signaling in vascular endothelial cells.

Proteinase-activated receptor 1 (PAR1) is a major signaling receptor for thrombin in vascular endothelial cells and it causes endothelial dysfunction, which ultimately leads to the early stage of vascular disease development. Targeting the thrombin-PAR1 signaling pathway is expected to be a potential strategy for preventing vascular diseases. The present study aimed to identify food-derived constituents that inhibit thrombin-PAR1 signaling. The extracts of the wine grape 'Kadainou R-1' (Vitis ficifolia var. ganebu × Vitis vinifera), were used as starting materials. The 50% ethanol extract significantly inhibited thrombin-induced, but not PAR1-activating peptide-induced, intracellular Ca2+ signaling in porcine aortic endothelial cells. The extract inhibited cleavage of the extracellular region of PAR1 by thrombin and the proteolytic activity of thrombin in an uncompetitive manner. The 50% ethanol extract contained polyphenols, including catechin, epicatechin, epicatechin gallate, resveratrol, oenin, and naringenin-7-O-glucoside, as major constituents. The removal of polyphenols by treatment with polyvinylpolypyrrolidone abolished approximately half of the inhibitory effect of the extract on the proteolytic activity of thrombin. Molecular docking simulation revealed the interaction of identified polyphenolic compounds with either exosite I, exosite II, or the catalytic site, depending on the model of the thrombin structure used. In conclusion, the 50% ethanol extract of wine grapes contains active constituents that inhibit the proteolytic activity of thrombin, thereby inhibiting PAR1-mediated Ca2+ signaling. Half of this inhibitory effect is attributable to polyphenolic compounds. These findings warrant the further exploration of natural compounds that target thrombin-PAR1 signaling.