Thrombin is a major physiological agonist that activates platelet secretion and aggregation. Nevertheless, our recent studies have shown that thrombin induces the development of apoptotic-like events in human platelets, mediated by endogenous H₂O₂ generation, including mitochondrial membrane potential depolarisation, cytochrome c release, caspase-3 and -9 activation and phosphatidylserine exposure. Proapoptotic proteins, such as Bid (a BH-3 only protein) and Bax (a critical multidomain downstream mediator of apoptosis), are key factors involved in the decrease of the mitochondrial membrane potential in nucleated cells. In the present study we have investigated the effect of thrombin on the activation and translocation from cytosol to mitochondria of Bid and Bax in human (non-nucleated) platelets. Subcellular fractionation was performed using a commercial Mitochondria Isolation Kit (Pierce Biotechnology, Inc., USA). The amount of proapoptotic proteins activated and their translocation were determined by immunoprecipitation and Western blotting using specific antibodies directed towards the active forms of Bid and Bax.

Platelets stimulation with thrombin (0.01-1 U/mL) for 1-60 min at 37ordm;C resulted in a concentration- and time-dependent activation and translocation from cytosol to mitochondria of Bid and Bax. The maximal activation (2-fold increase) was achieved after stimulation with 1 U/mL thrombin during 60 min. Platelet treatment with thrombin (1 U/mL) for 60 min decreased the amount of active proapoptotic proteins in the cytosolic fraction by 30 % whilst increased its amount in the mitochondrial fraction by 80 %.These findings suggest that proapoptotic proteins Bid and Bax are involved in thrombin-induced platelet apoptosis. Supported by MEC-DGI grant BFU2004-00165.