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 has 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 redox dependent regulation of platelets.

To begin, prepare the appropriate coating solution in modified Tyrode's buffer and coat an 8-well microslide by incubating it for 2 hours at 37 C.Then wash the coated microslide twice with modified Tyrode's buffer. Incubate the microslide in modified Tyrode's buffer containing 5 mg per mL BSA for at least 30 minutes to quench non-specific adhesion. To prepare washed platelets, centrifuge citrate-anticoagulated whole blood at 200 G for 20 minutes to obtain platelet-rich plasma.

Then add indomethacin and PGE1 to the platelet-rich plasma and centrifuge at 500 G for 10 minutes. Resuspend the resulting platelet pellet in modified Tyrode's HEPES buffer at 37 C to achieve a density of 4 x 10 to the power of 7 platelets per mL. Allow the isolated platelets to rest for 30 minutes at 37 C.Next, add DHE solution to the platelet suspension to achieve a final concentration of 10 M and incubate at 37 C for 1 minute.

After incubation, remove the blocking solution from the microslide and position it on the microscope stage for imaging. Then gently dispense the platelets. Use a 40x oil immersion lens on an inverted confocal microscope to monitor the conversion of DHE to 2-hydroxyethidium.

Image for 10 minutes, collecting images every 10 seconds. To monitor superoxide anion generation in response to an agonist, add a soluble agonist 10 minutes after dispensing the platelets and collect fluorescence images for an additional 10 minutes. Using this protocol, the generation of superoxide radicals was studied during platelet adhesion to collagen or fibrinogen, and from platelets adhered to PLL stimulated with thrombin.

To begin, obtain platelet-rich plasma by centrifuging the citrate-anticoagulated whole blood at 200 G for 20 minutes. Add indomethacin and PGE1 to the plasma and centrifuge at 500 G for 10 minutes. Resuspend the resulting platelet pellet in modified Tyrode's HEPES buffer at 37 C to obtain a density of 2 x 10 to the power of 8 platelets per mL.

Rest the platelets for 30 minutes at 37 C.Add DETC to a final concentration of 5 M and deferoxamine to a final concentration of 25 M into the platelet suspension followed by CMH to a final concentration of 200 M.Place the samples into aggregation cuvettes equipped with Teflon coated stirring magnets, and load them onto the aggregometer. After 1 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 microcentrifuge tube and quickly spin down at 6, 000 G for 10 seconds.

Load 50 L of the supernatant in capillary micropipettes 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.