![Figure 1. Intracellular ROS production in platelets and platelet sources of ROSs. (A) Washed human platelets (300 × 109/L) were preincubated with H2DCF-DA (50 μM) for 30 minutes, 37°C in phosphate-buffered saline (PBS)/5.5 mM glucose/1 mM EDTA (ethylenediaminetetraacetic acid), excessive dye was washed out, and platelets were resuspended in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer. Platelets were stimulated with thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), or ADP (50 μM) for 1 minute. Samples were then analyzed for intracellular ROS production by flow cytometry. In control experiments, ROS production was measured in neutrophils after PMA (0.1 μM) stimulation. Neutrophils produced high amounts of extracellular ROSs (420-fold increase versus control after 20 minutes of PMA stimulation) as measured by L-012. Intracellular production (H2DCF-DA, 10 μM) was much lower (PMA stimulation 6-fold increase versus control). In both cases, ROS production was inhibited by DPI and tiron. No signal for extracellular ROSs was observed in platelets in the same experiment (not shown). Data are represented as arbitrary units, mean ± SD from 5 experiments, control taken as 1 (*significantly different at P < .05 compared with control; #significantly different at P < .05 compared with PMA). (B) Washed human platelets (300 × 109/L) were preincubated with inhibitors of NAD(P)H oxidase (DPI [10 μM] or apocynin [600 μM]), mitochondrial metabolism (rotenone [100 μM]), xanthine oxidase (oxypurinol [100 μM]), COX (ASA 100 μM]), or NOS (nitro-l-arginine methyl ester [l-NAME]) for 5 minutes. Then, after activation by thrombin (0.2 U/mL) for 1 minute, ROS production was measured by flow cytometry. Platelet ROS production was also inhibited with intracellular superoxide scavengers tiron (3 mM) and MnTMPyP (100 μM) after thrombin stimulation. Data are shown as arbitrary units, mean ± SD, thrombin-induced ROS production taken as 1 (*significantly different at P < .05 compared with thrombin; n = 6). Error bars indicate 50 (from mean).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/106/8/10.1182_blood-2005-03-1047/6/zh80200585400001.jpeg?Expires=1724232258&Signature=fj2~HmHpEn-EUdxufZVtcmOTH2AusvSYsy3JpZdb-nhVF8oAjZr2-QTjGuKFCLyVS9M3FGoCDilHNlgoLThtOsMtsuzli27wHl6wcNK5GNAhiZKMI-a47nMkbbO7HbxoE2CgNxuHevkcfbsPL~Boji46vCVAlaGFFrlREoeYBN993WlNhxuMbA8MMr5hNYutMgbaBr~0vUQO2hczfOiF91MpKB-3Bcy4kc9T2tzbYQ8gGGONHudVJC63bhCjZ9lVdZKSi-Cn19KSkn-zlkqfgb2HRVKSC6sNOjdobiQROmA5hHHi-nXgIyLPDt3hxEzkCdnvHjVjiQvJp06jLjkXoQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Intracellular ROS production in platelets and platelet sources of ROSs. (A) Washed human platelets (300 × 109/L) were preincubated with H2DCF-DA (50 μM) for 30 minutes, 37°C in phosphate-buffered saline (PBS)/5.5 mM glucose/1 mM EDTA (ethylenediaminetetraacetic acid), excessive dye was washed out, and platelets were resuspended in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer. Platelets were stimulated with thrombin (0.2 U/mL), Trap6 (30 μM), U46619 (1 μM), convulxin (100 ng/mL), or ADP (50 μM) for 1 minute. Samples were then analyzed for intracellular ROS production by flow cytometry. In control experiments, ROS production was measured in neutrophils after PMA (0.1 μM) stimulation. Neutrophils produced high amounts of extracellular ROSs (420-fold increase versus control after 20 minutes of PMA stimulation) as measured by L-012. Intracellular production (H2DCF-DA, 10 μM) was much lower (PMA stimulation 6-fold increase versus control). In both cases, ROS production was inhibited by DPI and tiron. No signal for extracellular ROSs was observed in platelets in the same experiment (not shown). Data are represented as arbitrary units, mean ± SD from 5 experiments, control taken as 1 (*significantly different at P < .05 compared with control; #significantly different at P < .05 compared with PMA). (B) Washed human platelets (300 × 109/L) were preincubated with inhibitors of NAD(P)H oxidase (DPI [10 μM] or apocynin [600 μM]), mitochondrial metabolism (rotenone [100 μM]), xanthine oxidase (oxypurinol [100 μM]), COX (ASA 100 μM]), or NOS (nitro-l-arginine methyl ester [l-NAME]) for 5 minutes. Then, after activation by thrombin (0.2 U/mL) for 1 minute, ROS production was measured by flow cytometry. Platelet ROS production was also inhibited with intracellular superoxide scavengers tiron (3 mM) and MnTMPyP (100 μM) after thrombin stimulation. Data are shown as arbitrary units, mean ± SD, thrombin-induced ROS production taken as 1 (*significantly different at P < .05 compared with thrombin; n = 6). Error bars indicate 50 (from mean).
