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

Abstract

To cite this article: Lopez JJ, Salido GM, Gó mez-Arteta E, Rosado JA, Pariente JA. Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets. J Thromb Haemost 2007; 5: 1283-91.Summary. 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 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. Conclusion: Our results indicate that thrombininduced apoptosis is likely mediated by endogenous generation of H 2 O 2 in human platelets.

Sesamol induces apoptosis in human platelets via reactive oxygen species-mediated mitochondrial damage

“…Sesamol treatment induced a dose-dependent increase in ROS generation and it was found to be more potent than A23187 (positive control) at higher concentrations. Reports indicate that H 2 O 2 is the predominant ROS which stimulates the apoptotic events in platelets via the intrinsic pathway by altering the JDm [3]. Hence the endogenous generation of H 2 O 2 was also measured.…”

“…An ROS-sensitive fluorescent probe CMH2DCFDA was used to detect endogenous ROS production as described by Lopez et al [3], with slight modifications. Pre-treated (with 10 mg/mL calcium ionophore A23187 as standard agonist or with varying concentrations of sesamol as test group for 1 h at 37 C) and control (untreated) PRP as well as washed platelet suspensions were made up to a final volume of 200 mL with HEPES-buffered saline (HBS) HEPES-buffered saline (HBS), pH 7.45, containing 145 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 5 mM KCl, 1 mM MgSO 4 and supplemented with 0.1% Bovine Serum Albumin (BSA), incubated with 10 mM CMH2DCFDA in a polystyrene 96-well microtiter plate for 30 min at 37 C. Fluorescence was recorded with a Varioskan multimode plate reader (Thermo Scientifics, USA.)…”

“…Although platelets are anuclear, they do undergo apoptosis, a process of programmed cell death. The events of platelet apoptosis via the intrinsic pathway include only the cytoplasmic events initiated by the increased rate of endogenous reactive oxygen species (ROS) principally hydrogen peroxide (H 2 O 2 ) [3]. Apart from contributing towards the cell's energy metabolism, the role of mitochondria as pivotal mediators of intrinsic pathway of apoptosis is well documented.…”

Regulatory Role of Proteasome in Determination of Platelet Life Span

“…As membrane potential collapses, the fluorescence changes from red to green due to release of monomeric dye (16) Flow Cytometric Measurement of Reactive Oxygen Species (ROS)-Platelets were treated with proteasome inhibitors or vehicle as above, washed with PBS, and incubated with H 2 DCFDA (1 M) for 30 min at 37°C in the dark. Cells were next washed twice with PBS and analyzed by flow cytometry as described previously (16).…”

Propensity of crocin to offset Vipera russelli venom induced oxidative stress mediated neutrophil apoptosis: a biochemical insight

“…Immunobloting was carried out to detect the cyt-c release from cytosolic fractions of the samples (Lopez et al 2007). Cytosolic proteins were separated on 10 % SDS-PAGE and electrophoretically transferred on to a Polyvinylidene fluoride (PVDF) membrane for 1 h at 50 V using a wet blotter.…”