detection of superoxide anion in platelets using electron paramagnetic resonance

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

The superoxide anion (O2&#8226;-) is the most functionally relevant ROS generated in platelets1. O2&#8226;- is the product of the reduction of molecular oxygen and the precursor of many different ROS 2. The dismutation of O2&#8226;- leads to the generation of hydrogen peroxide (H2O2) via spontaneous reactions in aqueous solution or reactions catalyzed by superoxide dismutases (SODs3). Although different enzymatic sources have been suggested (e.g., xanthine oxidase4, lipoxygenase5, cyclooxygenase6, and nitric oxide synthase7), mitochondrial respiration8,9 and nicotinamide adenine dinucleotide phosphate-oxidases (NOXs)10 are the most prominent sources of superoxide anion in eukaryotic cells. This also seems to be the case in platelets, where electron leakage from mitochondrial respiration11,12 and the enzymatic activity of NOXs13,14 have been described as the main contributors to the superoxide anion output.
Although several studies have focused on the regulation of platelets by O2&#8226;-, there is no consensus regarding the molecular mechanisms responsible. The modulation of surface receptor activity via direct oxidation and disulfide bond formation has been proposed for different platelet receptors. The positive regulation of integrin &#945;IIb&#946;3 by ROS via direct oxidation of cysteine residues has been suggested15,16,17. Similarly, since platelet responses to collagen depend on disulfide-dependent dimerization and consequent dimerization of the glycoprotein VI (GPVI)18, receptor activity potentiation by ROS-dependent oxidation has been proposed19, although not fully proven experimentally. Finally, ROS-induced oxidation of the sulfhydryl groups of glycoprotein Ib (GPIb) was shown to promote platelet adhesion and platelet-leukocyte interaction during inflammation20. Conversely, as a possible consequence of decreased sulfhydryl group oxidation and receptor activation, the shedding of the ectodomain of both GPVI and GPIb is diminished&#160;by reducing conditions21.
Modes of action independently of a direct oxidation of platelet surface receptors have also been proposed. ROS, including O2&#8226;-, have been shown to positively modulate the collagen receptor GPVI by attenuating the activity of the Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2), which negatively regulates the signaling cascade of this receptor22. Moreover, O2&#8226;- can generate ONOO- (peroxynitrite) by rapid reaction with nitric oxide (NO), which normally inhibits platelets through the NO-sensitive guanylyl cyclase (NO-GC) and the generation of the negative platelet regulator cyclic GMP (cGMP)23,24. The resulting decrease in NO levels can lead to platelet potentiation. Alternatively, the generation of O2&#8226;- by NOX2 has been suggested to contribute to lipid peroxidation and isoprostane formation, which is essential for platelet activation and adhesion25. Finally, mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 5 (ERK5), a protein kinase proposed as a redox stress sensor in platelets26, is activated by O2&#8226;- and induces a procoagulant phenotype in platelets (as estimated by flow cytometry-based measurement of phosphatidylserine externalization)27.
The dysregulation of O2&#8226;- and other ROS generation in platelets has been associated with the exaggerated hemostatic response leading to thrombotic cardiovascular complications associated with atherosclerosis, diabetes mellitus, hypertension, obesity, and cancer28,29. In these pathological settings, the ROS output by platelets is increased, which leads to a potentiation of their adhesive and aggregatory responses. In addition to the effect on platelet responses, the free radical output of platelets may have consequences on other blood cells and vascular structures, which is a poorly understood and underinvestigated area of cardiovascular health30. Despite our limited understanding of the molecular mechanisms linking oxidative stress to thrombotic conditions, the clinical relevance of antioxidants for the protection against cardiovascular disease has received considerable attention. Plasma antioxidant levels have been shown to inversely correlate with the risk of developing cardiovascular conditions, and dietary antioxidant consumption has been shown to protect against coronary artery disease31,32.&#160;Consequently, the use of dietary antioxidants has been advocated as a promising approach for cardiovascular disease prevention33,34,35. Amongst the effects of ROS generation in platelets, the increase in apoptosis can have important pathophysiological effects36,37. Overall, reliable protocols to detect and quantify the O2&#8226;- output by platelets are increasingly relevant in cardiovascular research.
Currently, available techniques for the detection of ROS have important limitations of specificity (i.e., the chemical nature of the oxidant molecules detected is unknown) and reliability (i.e., the unwanted interaction with biological molecules and experimental reagents leads to biased non-physiological results)38,39. The most commonly used approach for the detection of ROS in platelets is based on the use of dichlorodihydrofluorescein diacetate (DCFDA), which is converted to dichlorodihydrofluorescein (DCFH) by intracellular esterases and consequently to the highly fluorescent dichlorofluorescein (DCF) by cellular oxidants, including hydroxyl radicals and peroxidase-H2O2 intermediates40,41. Despite its wide use, serious questions have been raised regarding the reliability of this approach for the measurement of intracellular ROS38. The oxidation of DCFH to DCF can be, in fact, induced by transition metal ions (e.g., Fe2+) or heme-containing enzymes (e.g., cytochromes) instead of ROS42. Moreover, DCFDA is converted by cell peroxidases to its semiquinone free radical form (DCF&#8226;-), which is in turn oxidized to DCF by reaction with molecular oxygen (O2) with the release of O2&#8226;-, which leads to the artificial amplification of oxidative responses41,43,44. Therefore, the detection of intracellular ROS by DCFDA is useful for obtaining initial insights but requires cautious consideration and extensive experimental controls38,39.
This study presents three alternative techniques for the detection and measurement of the key regulator of platelet function O2&#8226;-1. The first technique is the detection using DHE and flow cytometry, which offers advantages of reliability and specificity over DCFDA. The second technique proposed here also utilizes DHE, but the detection method is live-platelet fluorescence imaging, which allows the study of the generation of O2&#8226;-&#160;upon platelet signaling with fast kinetics and single-cell resolution. Finally, a protocol based on the use of the hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) in EPR resonance experiments offers the possibility of quantifying the rate of O2&#8226;- generation by platelets and comparing it in different conditions.

Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets 

The scope of our research is to understand how the generation of reactive oxygen species, regulates platelet function and modulates the hemostatic response. For our work, it is essential to reliably detect the key reactive oxygen species, superoxide anion. The most common technique to detect superoxide anion is flow cytometry with DCFDA as fluorescent probe.These as limitations, it induces the release of reactive oxygen species leading to unreliable data. It does not allow to distinguish, between different reactive oxygen species, and it does not allow to quantify platelet reactive oxygen species. This video presents two alternative techniques for the detection of platelet superoxide anions.The first technique utilizes DHE on live platelets to detect superoxide anion with fast kinetics and single-cell resolution. The second technique utilizes the spin probe, CMH, to quantify the rate of superoxide anion generation by platelets. Overall, the techniques presented in this video, offer significant advantages over most existing techniques, and represent a significant advance in the experimental tools at our disposal to study the redux-dependent regulation of platelets.

Detection Of Superoxide Anion in Platelets Using Electron Paramagnetic Resonance

To begin, obtain platelet-rich plasma by centrifuging the citrate anticoagulated whole blood at 200g for 20 minutes. Add indomethacin and PGE1 to the plasma and centrifuge at 500g for 10 minutes. Resuspend the resulting platelet pellet in modified Tyrode's HEPES buffer at 37 degrees Celsius to obtain a density of 2x10^8 platelets per milliliter.Rest the platelets for 30 minutes at 37 degrees Celsius. Add DETC to a final concentration of five micromolar and deferoxamine to a final concentration of 25 micromolar into the platelet suspension, followed by CMH to a final concentration of 200 micromolar. Place the samples into aggregation cuvettes equipped with Teflon-coated stirring magnets, and load them onto the aggregometer.After one minute, add stimuli such as thrombin or collagen, and incubate for 10 minutes. After incubation, transfer the platelet suspension from the aggregation cuvette to a micro centrifuge tube and quickly spin down at 6, 000g for 10 seconds. Load 50 microliters of the supernatant in capillary micro pipettes and seal with EPR sealing wax.Transfer samples to the EPR scanner and set the parameters on the scanner. Estimate CMH oxidation to CM radical as the area under the curve of the recorded EPR peaks. Obtain a calibration curve using commercially available CM radical.The EPR signal intensity in the samples was proportional to the intensity of the EPR peaks. The specificity of detection was confirmed with ROS scavengers and selective inhibitors of enzymes generating superoxide anion. Data for platelet stimulation with thrombin or collagen in the presence of the selective inhibitor VAS2870 suggested that NADPH oxidases are the main source of platelet superoxide anions in response to both these agonists.

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Research

JoVE Journal

Immunology and Infection

21 Views. To begin, obtain platelet-rich plasma by centrifuging the citrate anticoagulated whole blood at 200g for 20 minutes. Add indomethacin and PGE1 to the plasma and centrifuge at 500g for 10 minutes. Resuspend the resulting platelet pellet in modified Tyrode's HEPES buffer at 37 degrees Celsius to obtain a density of 2x10^8 platelets per milliliter.Rest the platelets for 30 minutes at 37 degrees Celsius. Add DETC to a final concentration of five micromolar and deferoxamine to a final conce...