A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β

Abstract

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 μg/ml CRP and 30 μM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid β (Aβ) 1-42 peptide induced superoxide anion formation in a concentration-dependent manner. Aβ peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aβ 1-42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 μM arachidonic acid. The inhibition of NOXs by 10 μM VAS2870 abolished Aβ-dependent potentiation of platelet aggregation in response to 10 μM arachidonic acid, suggesting that the pro-thrombotic activity of Aβ peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aβ peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets.

NADPH oxidase 1 is a novel pharmacological target for the development of an antiplatelet drug without bleeding side effects

“…Using washed platelets from NOX1 −/− and NOX2 −/− transgenic mice, we have also proved that NOX1 is critical for collagen responses, while the latter is required for thrombin responses. This is in agreement with previous studies, 10,13,14 but in contrast with other reports. 9,37 This discrepancy is difficult to explain, although it can be at least partially explained with the differences in experimental tools and conditions utilized.…”

“…13,24 Recently, we utilized selective agonists and a novel electron paramagnetic resonance-based technique to show that the signaling of the collagen receptor GPVI requires NOX1. 14 These results are paralleled by a recent study showing that NOX2 is dispensable for GPVI-dependent activation of platelets ex vivo and the carotid thrombosis induced by photochemical injury in vivo. 12 This recent evidence is in contrast with previous studies indicating NOX2 as critical for the activation of platelets by collagen and for the development of thrombosis in vivo.…”

“…As described in our previous study, 14 prior to adding stimuli, 200 µM 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), 5 µM diethyldithiocarbamate (DETC), and 25 µM deferoxamine were added to platelets (density adjusted to 2 × 10 8 platelets/mL) with continuous stirring. After 1 minute, stimuli were delivered and 10 minutes later 50 μL of platelet-free supernatant was collected and read using an E-scan (Noxygen, Germany).…”

“…10 Despite the established importance of NOXs in the molecular mechanisms leading to platelet activation, 8,9,11 the exact role of different NOX isoenzymes, and the molecular mechanism linking NOX activity to platelet regulation remains uncertain. 9,[12][13][14] Nonetheless, a convincing amount of evidence suggests that the enzymatic activity of NOXs plays a significant role in promoting platelet responses. 15 Therefore, several pharmacological tools for the inhibition of NOXs that have been recently developed and described may have important platelet modulatory effects.…”

“…NOX1 selectivity of 2-acetylphenothiazine (2APT) was then confirmed using electron paramagnetic resonance (EPR) spectroscopy. 14 The calibration curve utilized to quantify the superoxide anion output is shown in Figure S1. In these experiments, 2APT significantly inhibited the superoxide anion generation induced by collagen on wild-type platelets, but not in NOX1 −/− platelets ( Figure 3A).…”