Plant Food Anthocyanins Induced Platelet Apoptosis Via BCL-2/BCL-XL Pathway

Background: Platelets are versatile cells and play important roles in hemostasis/thrombosis, inflammation, and atherosclerosis. The pathogenesis of cardiovascular diseases (CVDs) is linked to platelet hyperactivity which is considered an independent risk factor for CVDs. Platelets are critical for promoting the progression of CVDs, and platelet apoptosis have been reported to be involved in platelet activation. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies demonstrated that anthocyanins from plant food significantly inhibited platelet activation, adhesion, aggregation and granule secretion, as well as attenuated thrombus growth at both arterial and venous shear stresses in vitro and in vivo, however, the effects of anthocyanin on platelet apoptosis and its mechanisms have not been explored. In the present study, we examined whether anthocyanin Cyanidin-3-glucoside (Cy-3-g) affect platelet apoptosis and the BCL-2/BCL-XL intrinsic apoptotic pathway.

Methods: Cy-3-g, the predominant bioactive compound of anthocyanin preparations, was obtained from Polyphenol AS Company in Norway.Purified gel-filtered platelets from healthy volunteers were incubated at 37^oC for 40 minutes with different concentrations of Cy-3-g (0.5、5、50μM) or PBS buffer as a control. the activated platelets were triggered with 0.5U thrombin for 15min to induce apoptosis. Mitochondria membrane potential (Δψm) and membrane phospholipid phosphatidylserine (PS) exposure in both activated and resting platelets were assessed by flow cytometry. Cytochrome C release, activation of caspase-3, caspase-8, caspase-9, cleavage of gelsolin, the levels of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and proapoptotic BCL-2 family proteins Bax, Bak, Bad, Bid and tBid in both activated and resting platelets were measured by western blotting.

Results: Cy-3-g at 5μM and 50μM directly induced significant ΔΨm dissipation in activated platelets dose dependently. Correspondingly, 50μM Cy-3-g increased cytochrome C release compared to control. The expression of pro-caspase-8 and pro-caspase-9 decreased, activation of caspase-3, caspase-8 and caspase-9 was induced in activated platelets in both 5μM and 50μM Cy-3-g groups. Both PS exposure and the cleavage of gelsolin increased in activated platelets, however these effects were only observed at Cy-3-g doses as high as 50μM. Cy-3-g did not induce the above changes in resting platelets. The intrinsic apoptotic pathway was initiated by Cy-3-g treatment in activated platelets; Cy-3-g significantly inhibited the expression of BCL-2, BCL-XL and increased the levels of Bax, Bak, Bad and Bid in activated platelets dose dependently. No significant difference was observed in resting platelets.

Conclusions: Our data demonstrate for the first time that purified anthocyanin Cy-3-g directly accelerated apoptosis in activated platelets via the BCL-2/BCL-XL pathway. Anthocyanins may possess therapeutic potential for patients suffering from thrombotic conditions.