A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli

Abstract

The regulation of platelets by oxidants is critical for vascular health and may explain thrombotic complications in diseases such as diabetes and dementia, but remains poorly understood. Here, we describe a novel technique combining electron paramagnetic resonance spectroscopy and turbidimetry, which has been utilized to monitor simultaneously platelet activation and oxygen radical generation. This technique has been used to investigate the redox-dependence of human and mouse platelets. Using selective peptide inhibitors of NADPH oxidases (NOXs) on human platelets and genetically modified mouse platelets (NOX1 −/− or NOX2 −/− ), we discovered that: 1) intracellular but not extracellular superoxide anion generated by NOX is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent activation; 3) NOX1 is the main source of oxygen radicals in response to collagen, while NOX2 is critical for activation by thrombin; 4) two platelet modulators, namely oxidized low density lipoproteins (oxLDL) and amyloid peptide β (Aβ), require activation of both NOX1 and NOX2 to pre-activate platelets. This study provides new insights into the redox dependence of platelet activation. It suggests the possibility of selectively inhibiting platelet agonists by targeting either NOX1 (for collagen) or NOX2 (for thrombin). Selective inhibition of either NOX1 or NOX2 impairs the potentiatory effect of tested platelet modulators (oxLDL and Aβ), but does not completely abolish platelet hemostatic function. This information offers new opportunities for the development of disease-specific antiplatelet drugs with limited bleeding side effects by selectively targeting one NOX isoenzyme.

Fibrillar amyloid peptides promote platelet aggregation through the coordinated action of ITAM‐ and ROS‐dependent pathways

“…36 By contrast, the few available studies using the full peptides Aβ40 and Aβ42 are incomplete and have generated controversial results on their effective ability and potency to trigger platelet responses. 11,14,15,19 The current incompleteness and incongruences are reasonably because amyloid peptides tend to form fibrils in aqueous solutions, with specific intrinsic kinetics, and the implications of their biochemical conformation on biological activities was not properly considered. Amyloid Aβ fibrils are present in atherosclerotic plaques, and Aβ peptides are also detected in the circulation.…”

“…Previous studies had reported that Aβ42, but not Aβ40, promotes the formation of ROS in platelets, through the activation of NADPH oxidase NOX1 and NOX2. 14,15 However, in these studies, the chemical form of the peptides was not characterized. Therefore, we recon- Intracellular ROS production was then evaluated in flow cytometry, as described in the Methods.…”

“…[9][10][11][12][13] Moreover, in line with their ability to stimulate oxidative burst in neutrophils, Aβ peptides trigger the generation of reactive oxygen species (ROS) in platelets in a NOX-dependent manner. [14][15][16] The mechanism of platelet activation by Aβ peptides is controversial. Some studies have indicated that Aβ peptides interact with different membrane receptors, including APP itself, PAR1, CD36, GPIb-IX-V complex, or integrin αIIbβ3, but their involvement in the initiation of platelet activation is uncertain.…”

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

“…[1][2][3][4] NADPH oxidases (NOXs) are important sources of ROS in platelets [5][6][7] and their function promotes platelet activation. 8,9 NOXs are a family of multimeric plasma membrane complexes generating the majority of non-mitochondrial ROS in mammalian cells. Each complex consists of a catalytic subunit and a number of regulatory subunits responsible for the maturation, stabilization, haem incorporation, and translocation to the cell membrane of the enzymatically functional complex.…”

“…Although platelet NOX2 has been investigated, 4 the expression of NOX1 in human platelets has only been described relatively recently. 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.…”