Journal
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Volume 5, Issue 6, Pages 1283-1291Publisher
WILEY
DOI: 10.1111/j.1538-7836.2007.02505.x
Keywords
apoptosis; caspase-3; caspase-9; hydrogen peroxide; platelets; thrombin
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Background: Thrombin is a major physiological platelet agonist that activates a number of cell functions including aggregation. Platelet stimulation with thrombin has been shown to result in the development of apoptotic events, including activation of caspases-3 and -9, cytochrome c release and phosphatidylserine (PS) exposure; however, the mechanism underlying the activation of apoptosis remains unclear. Objectives: In the present study, we aim to investigate whether endogenously generated reactive oxygen species upon thrombin stimulation is required for the activation of apoptosis in human platelets. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC-1; caspase-3 and -9 activity was determined from the cleavage of their respective specific fluorogenic substrates; PS externalization was estimated using annexin V-fluorescein isothicyanate and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin stimulates mitochondrial membrane potential depolarization and endogenous generation of H2O2. Platelet exposure to exogenous H2O2 results in cytochrome c release and activation of caspases-9. In addition, H2O2 induces the activation of caspase-3 and PS exposure by a mechanism dependent on cytochrome c release and caspase-9 activation. Finally, thrombin-evoked development of apoptotic events was impaired by treatment with catalase. Conclusion: Our results indicate that thrombin-induced apoptosis is likely mediated by endogenous generation of H2O2 in human platelets.
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