Potent and specific effects of Grape Seed extract on platelet aggregation.

The beneficial anti-platelet effects of red wine drinking have been widely reported, and have largely been credited to the grape seeds and skins being an excellent source of flavonoids. However, there is little understanding of the mechanism by which flavonoids act on platelets. This study investigated changes in aggregation in response to ADP and phorbol myristate acetate (PMA) induced by grape seed extract (GSE). Venous blood (50 ml) was obtained from healthy subjects who were not taking any medication and had refrained from flavonoid consumption that day. Whole blood was mixed with 10% volume of 3.2% sodium citrate to prevent clotting, and the plasma rich platelet (PRP) fraction was isolated by centrifugation at 1,100 rpm for 15 mins. Platelet poor plasma (PPP) was prepared by centrifugation of the PRP at 2000 rpm for 5 mins. Platelet aggregation was performed using a dual chamber Payton aggregation model and the instrument calibrated for each individual using the PRP compared to PPP fractions. PRP samples were incubated with 10 μg/ml GSE or vehicle for 4 minutes prior to aggregation with an EC50 of either ADP or PMA. The extracts were dissolved in vehicle consisting of: 2% ethanol, 1 mM citric acid, 5 mM HCl in 5 mM ascorbic acid. ADP induces a biphasic aggregation response; the initial phase was not affected by the presence of either extract. However, the second phase was significantly inhibited by pre-incubation with GSE (74.3 ± 13.4%; n  =  3) compared with vehicle control. PMA induced aggregation was not changed by incubation with either extract. This data not only demonstrates that grape extracts are potent inhibitors of aggregation ex vivo, but also suggests that flavonoids may act on the PLC second messenger pathway.