Ethanol Induces Platelet Apoptosis

【Background】Alcoholic beverage is one of the most favorite drinks in daily life. However, alcohol abuse has become a common social problem around the world. Alcohol abuse incurs severemedial conditions, such as thrombocytopenia and hemorrhage, but the pathogenesis is not totally understood.【Methods】Fresh blood from health volunteers aged 18-25 years old without taking any medication within two weeks were collected and centrifugated, then washed platelets and platelet rich plasma(PRP) were prepared. Washed platelets(3 × 108/ml) were incubated with different concentrations of ethonal or vehicle dimethyl sulfoxide(DMSO) at 37°C for 2 hours. Then, mitochondrial inner transmembrane potential(ΔΨm), P-selection and PAC-1 binding were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein(Bax), and anti-apoptotic proteins(Bcl-2). Activation of caspase-3 and c-jun NH2-terminal kinase(JNK) were also examined by western blot. To test whether inhibition of JNK can attenuate ethonal-induced platelet apoptosis, the treated platelets were analyzed by western blot for the expression phosphorylation levels of JNK protein. For platelet aggregation analysis, PRP was incubated with ethonal(200 mg/dl) or vehicle control(DMSO) at 37 ℃ for 2 hours, washed platelets were incubated with ethonal(200 mg/dl) or vehicle control(DMSO) at 37 ℃ for 2 hours. Platelet aggregation assay were performed by addition of adenosine diphosphate(ADP, 10 μmol/L) into PRP, or thrombin(0.5 U/m L),collagen(5 μg/m L) into washed platelets at 37 ℃, and examined by a turbidometric platelet aggregometer(Chrono-log, PA, USA) at a stirring speed of 1000 rpm. In in vivo experiments, C57BL/6j mice were given ethanol by gavage, the numbers of circulating platelets were counted by Sysmex KX-21 N Blood Cell Analyser, the tail bleeding time and the stomach were examined.【Results】Ethanol dose-dependently induces depolarization of mitochondrial inner transmembrane potential, up-regulation of Bax,down-regulation of Bcl-2, and caspase-3 activation. Ethanol does not induce surface expression of P-selectin or PAC-1 binding, whereas, obviously reduces collagen, thrombin, and ADP-induced platelet aggregation. Moreover, ethanol induces c-Jun NH2-terminal kinase activation. In an in vivo mouse model of the acute alcoholism, ethanol obviously reduces the number of circulating platelets, prolongs the tail bleeding time, and incurs gastric mucosa hemorrhage.【Conclusions】These data demonstrate that ethanol incurs mitochondria-mediated intrinsic platelet apoptosis, results in the reduction of the number of circulating platelets and impairs in vivo hemostasis. These findings reveal the possible pathogenesis of hemorrhagic symptoms in patients experiencing acute alcohol intoxication.