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Platelet Biology & its Dsorders
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
Ferrata Storti Foundation
Haematologica 2019 Volume 104(9):1879-1891
Dina Vara,1 Eugenia Cifuentes-Pagano,2 Patrick J. Pagano2 and Giordano Pula1
1Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK and 2Department of Pharmacology and Chemical Biology and Vascular Medicine Institute, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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 gen- erated by NOX is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent acti- vation; 3) NOX1 is the main source of oxygen radicals in response to colla- gen, while NOX2 is critical for activation by thrombin; 4) two platelet mod- ulators, 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 complete- ly abolish platelet hemostatic function. This information offers new oppor- tunities for the development of disease-specific antiplatelet drugs with lim- ited bleeding side effects by selectively targeting one NOX isoenzyme.
Introduction
Platelets are anucleated circulating cells responsible for initiating hemostasis via thrombus formation and blood clotting. The regulation of platelets is of primary importance for cardiovascular medicine and for the discovery of new drugs to treat cardiovascular diseases.1 In addition to canonical signaling pathways depending on protein kinase activity,2 platelets are regulated in a redox-dependent manner. Several lines of evidence suggest that platelets are modulated by extracellular reactive oxygen species (ROS)3 and that platelet activation is essentially dependent on the generation of endogenous ROS.4-6 Therefore, the study of platelet regulation and hemostasis is shedding light on the interface between ROS biochemistry and cellular physiology.
•−
Superoxide anion (O2 ) from exogenous sources or endogenously produced by
platelets is shown to significantly increase platelet aggregation and thrombus forma-
7 •−
tion. O2 has a pre-eminent role in biology and pathophysiology, as it serves as a
progenitor for formation of hydrogen peroxide (H2O2), peroxynitrite (ONOO•−) and hydroxyl radical (HO•), and thereby plays a key role in the post-translational oxida- tive modification of proteins.8 The work of several research groups has focused on
Correspondence:
GIORDANO PULA
g.pula@exeter.ac.uk
Received: October 8, 2018. Accepted: January 18, 2019. Pre-published: January 24, 2019.
doi:10.3324/haematol.2018.208819
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/9/1879
©2019 Ferrata Storti Foundation
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haematologica | 2019; 104(9)
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