Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC131

Poster Communications

Activation and translocation of Bid and Bax to the mitochondria in response to thrombin in human platelets

J. J. Lopez1, G. M. Salido1, J. A. Pariente1, J. A. Rosado1

1. Department of Physiology, University of Extremadura, Caceres, Spain.


Apoptosis or programmed cell death is a well-conserved physiological pathway where the pro-apoptotic proteins Bid and Bax play an essential role. Bid or Bax translocate to mitochondria in response to apoptotic stimuli to initiate the release of cytochrome c. (Luo et al., 1998). Thrombin is a physiological platelet agonist that activates a number of cell functions, as well as apoptotic events, including cytochrome c release and subsequent phosphatidylserine exposure (Lopez et al., 2007). The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid and Bax and its relevance in thrombin-evoked apoptotic events. Blood was drawn from volunteers with local ethical committee approval and in accordance with the Declaration of Helsinki. Changes in the mitochondrial membrane potential were registered using the dye JC-1. Bid and Bax activation and translocation was detected by immunoprecipitación and Western blotting in whole cell lysates and samples from the mitochondrial and cytosolic fractions. Phosphatidylserine externalization was estimated using annexin V-FITC as previously shown (Lopez et al., 2007). Treatment of platelets with 1 U/mL thrombin induces concentration- and time-dependent activation and mitochondrial association of active Bid and Bax (thrombin enhanced the amount of active Bid and Bax in the mitochondria by 212 ± 25% and 200 ± 22% of control, respectively, mean ± S.E.M.; P<0.05, Student's t-test; n=6). Translocation of Bid and Bax to the mitochondria was reduced by treatment for 40 min with the actin polymerization inhibitor cytochalasin D (10 µM) to 131 ± 9% and 115 ± 11% of control, respectively, (P<0.05, Student's t-test; n=6). Blockage of the Bax channel significantly reduced thrombin-evoked mitochondrial membrane depolarization and phosphatidilserine exposure wihout having any effect on Bax activity. Our results indicate that thrombin induces activation and mitochondrial translocation of Bid and Bax, a process that is dependent on actin filament polymerization. Thrombin-evoked Bax channel formation is likely one of the initial apoptotic events in human platelets.

Where applicable, experiments conform with Society ethical requirements