Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets.
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.


OBJECTIVE
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 H(2)O(2) . Platelet exposure to exogenous H(2)O(2) results in cytochrome c release and activation of caspases-9. In addition, H(2)O(2) 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.


CONCLUSIONS
Our results indicate that thrombin-induced apoptosis is likely mediated by endogenous generation of H(2)O(2) in human platelets.
Lopez
J J
JJ
Department of Physiology (Cellular Physiology Research Group), University of Extremadura, Cáceres, Spain.
Salido
G M
GM
Gómez-Arteta
E
E
Rosado
J A
JA
Pariente
J A
JA
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D053148:Caspase 3 / Q000097:blood; D053453:Caspase 9 / Q000097:blood; D002374:Catalase / Q000494:pharmacology; D045304:Cytochromes c / Q000097:blood; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010718:Phosphatidylserines / Q000097:blood; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D002374:Catalase; D013917:Thrombin; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9