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D. Vara et al.
NADPH oxidases (NOX) as important sources of ROS in platelets responsible for the regulation of platelet respon- siveness.9-15 Despite the increased interest in this aspect of platelet biology and hemostasis regulation, progress within this field is hampered by the lack of reliable and quantita- tive techniques for the analysis of platelet oxidative sta- tus.16,17 This has made it challenging to completely appreci- ate the importance of endogenous and exogenous oxidants on the regulation of platelet signaling pathways and on the balance between hemostasis and thrombosis in health and disease. Indirectly, this has impeded the development of pharmacological treatments for thrombotic conditions based on the control of ROS generation. We addressed this biomedical need by combining the measurement of platelet activation (using turbidimetry18) and the simultaneous measurement of intracellular or extracellular oxygen radi- cals [using electron paramagnetic resonance (EPR) or EPR spectroscopy19,20] into one multiplex technique that allows the accurate study of the oxidative status and function of human platelets.
This technique is likely to find application in clinical prac- tice, where the simultaneous analysis of platelet responsive- ness and oxidative stress can help develop more advanced diagnostics for patients at risk of thrombotic diseases. It could also find application in drug discovery, as NOX mod- ulation is becoming an important therapeutic strategy in several diseases.21,22 In the cardiovascular field, in order to avoid side effects and bleeding complications of antithrom- botic drugs, modern drug discovery aims to develop target- ed approaches that interfere with the contribution of platelets to pathological alterations of the vascular system while preserving their vascular protective functions.23,24 Within this context, it is imperative to deepen our under- standing of the regulation of platelets in both health and disease, as redox-dependent regulation of platelets remains poorly understood.25 Our novel approach can help to clarify redox-dependent mechanisms regulating platelets and hemostasis, validate new drug discovery targets, and iden- tify novel antiplatelet drug candidates.
In this study, we utilized the EPR/turbidimetry technique to clarify the dynamics of the generation and activation of oxygen radicals in human platelets in response to physio- logical and pathological stimuli. The use of NOX1- and NOX2-selective peptide inhibitors allowed the identifica- tion of key differences in the involvement of these two enzymes in the response to platelet agonists and modula- tors. The application of this technique will further our understanding of redox-dependent platelet regulation and may have important consequences for antiplatelet drug dis- covery, where the quest for truly pathway-specific inhibitors targeting pathological platelet activation without interfering with their physiological hemostatic function remains an unmet objective.
Methods
Platelet preparation
Human blood was drawn from healthy volunteers by median cubital vein venepuncture following Royal Devon and Exeter NHS Foundation Trust Code of Ethics and Research Conduct and under National Research Ethics Service South West – Central Bristol approval (Rec. n. 14/SW/1089). Sodium citrate was used as antico- agulant (0.5% w/v). Platelet rich plasma (PRP) was separated from
whole blood by centrifugation [250xg, 17 minutes (min)], and platelets were separated from PRP by a second centrifugation step (500xg, 10 min), in the presence of prostaglandin E1 (PGE1, 40 ng/mL) and indomethacin (10 μM). For mouse platelets, blood was taken with intracardiac puncture from 12-week old females under the Home Office license PPL30/3348 and anticoagulated with 0.5% w/v citrate. PRP was separated from whole blood by cen- trifugation (160xg, 15 min), and platelets were separated from PRP by a second centrifugation step (600xg, 10 min), in the presence of prostaglandin E1 (PGE1, 40 ng/mL) and indomethacin (10 μM).
Electron paramagnetic resonance/turbidimetry assay
2x108 platelets/mL were prepared as described above. Before adding stimuli, 200 μM CMH or PPH was added to platelets in the presence of 25 μM deferroxamine and 5 μM diethyldithiocarba- mate (DETC). Platelet suspensions were loaded onto a Chronolog 700-2 aggregometer with continuous stirring and the turbidimetry readings were immediately started. 50 μL of platelet-free super- natant were transferred into the Hirschmann precision micropipettes and read using an e-scan (Noxygen, Germany).
Thrombus formation under physiological flow assay
The Bioflux200 system (Fluxion, South San Francisco, CA, USA) was used to analyze thrombus formation in human and mouse whole blood under flow. Heparin-anti- coagulated whole blood was anticoagulated with 5 μ/mL heparin plus 40 μM D-Phenylalanyl-prolyl-arginyl Chloromethyl Ketone or PPACK and incubated with scram- bled or the NOX inhibitory peptides, NoxA1ds and Nox2ds-tat before the addition of 1 μM 3,3'-dihexyloxacar- bocyanine iodide (DiOC6) for 10 min before the blood was added to the wells. Thrombus formation was visualized by fluorescence microscopy at a shear rate of 1000 sec−1.
A more detailed description of the methods used is avail- able in the Online Supplementary Appendix.
Results
Superoxide anion-dependence of platelet activation by collagen but not thrombin
Generation of ROS in a living cell can be examined by EPR spectroscopy using 1-hydroxy-3-methoxycarbonyl- 2,2,5,5-tetramethylpyrrolidine (CMH), an oxygen radical- specific spin probe.16 This spin probe crosses the plasma membrane and directly reacts with intracellular oxygen rad- icals to generate a nitroxide adduct that can be detected by EPR spectroscopy (Online Supplementary Figure S1A and B). We combined classical aggregometry (also known as tur- bidimetry) with CMH-dependent and EPR spectroscopy by analyzing CMH oxidation in the platelet supernatant while the aggregation reaction is taking place (Online Supplementary Figure S1C). EPR is considered a gold stan- dard for ROS detection and offers the important advantage of providing a quantification of the generation rate of oxy- genradicals.Itisinfactpossibletobuildacalibrationcurv●e using known concentrations of the nitroxide adduct (CM ) (Online Supplementary Figure S2A and B). Once platelet sus- pension density and incubation time is known, it is possi- ble to interpolate experimental data of resonance intensity to determine CMH oxidation rates (moles per platelet per min; see formula in Online Supplementary Figure S2C). This assay allowed us to correlate platelet aggregation induced by collagen and thrombin with the rate of oxygen radical generation (measured as rate of CMH oxidation). Collagen
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haematologica | 2019; 104(9)