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.
