DHE oxidation products in resting, activated, and aggregated platelets
| Nox2 genotype . | Thrombin and convulxin . | Analyte (pmol)/protein (µg) . | ||||
|---|---|---|---|---|---|---|
| 2-OH-E+ . | DHE . | E+ . | E+ dimer . | DHE dimer . | ||
| Pellet (intracellular) | ||||||
| +/y | — (RP) | 0.24 ± 0.04 | 0.55 ± 0.06 | 1.09 ± 0.05 | 0.24 ± 0.03 | 2.67 ± 0.11 |
| −/y | — (RP) | 0.23 ± 0.06 | 0 | 1.11 ± 0.09 | 0.30 ± 0.06 | 2.03 ± 0.19 |
| +/y | + (Act) | 0.25 ± 0.04 | 0.48 ± 0.06 | 0.80 ± 0.03 | 0.19 ± 0.02 | 1.80 ± 0.10 |
| −/y | + (Act) | 0.28 ± 0.06 | 0.59 ± 0.11 | 0.97 ± 0.07 | 0.12 ± 0.03 | 1.80 ± 0.18 |
| +/y | + (Agg) | 0.47 ± 0.05* | 1.16 ± 0.10 | 1.63 ± 0.07 | 0.68 ± 0.05 | 3.93 ± 0.15 |
| −/y | + (Agg) | 0.45 ± 0.08* | 0.57 ± 0.11 | 1.21 ± 0.09 | 0.47 ± 0.06 | 2.61 ± 0.21 |
| Supernatant (extracellular) | ||||||
| +/y | — (RP) | 5.4 ± 0.8 | 58.6 ± 4.3 | 45.8 ± 2.9 | 2.0 ± 0.7 | 34.0 ± 0.7 |
| −/y | — (RP) | 5.1 ± 1.7 | 57.5 ± 6.9 | 52.0 ± 5.0 | 2.1 ± 1.15 | 33.7 ± 0.3 |
| +/y | + (Act) | 5.2 ± 1.1 | 50.6 ± 5.8 | 38.3 ± 3.8 | 1.8 ± 0.4 | 33.8 ± 0.6 |
| −/y | + (Act) | 5.4 ± 1.6 | 50.9 ± 4.6 | 47.4 ± 5.4 | 1.9 ± 1.0 | 34.0 ± 0.4 |
| +/y | + (Agg) | 5.1 ± 0.7 | 72.0 ± 4.5 | 44.4 ± 2.9 | 1.2 ± 0.7 | 23.0 ± 6.0 |
| −/y | + (Agg) | 5.1 ± 1.1 | 65.0 ± 5.4 | 50.2 ± 4.2 | 3.6 ± 2.3 | 0 |
| Nox2 genotype . | Thrombin and convulxin . | Analyte (pmol)/protein (µg) . | ||||
|---|---|---|---|---|---|---|
| 2-OH-E+ . | DHE . | E+ . | E+ dimer . | DHE dimer . | ||
| Pellet (intracellular) | ||||||
| +/y | — (RP) | 0.24 ± 0.04 | 0.55 ± 0.06 | 1.09 ± 0.05 | 0.24 ± 0.03 | 2.67 ± 0.11 |
| −/y | — (RP) | 0.23 ± 0.06 | 0 | 1.11 ± 0.09 | 0.30 ± 0.06 | 2.03 ± 0.19 |
| +/y | + (Act) | 0.25 ± 0.04 | 0.48 ± 0.06 | 0.80 ± 0.03 | 0.19 ± 0.02 | 1.80 ± 0.10 |
| −/y | + (Act) | 0.28 ± 0.06 | 0.59 ± 0.11 | 0.97 ± 0.07 | 0.12 ± 0.03 | 1.80 ± 0.18 |
| +/y | + (Agg) | 0.47 ± 0.05* | 1.16 ± 0.10 | 1.63 ± 0.07 | 0.68 ± 0.05 | 3.93 ± 0.15 |
| −/y | + (Agg) | 0.45 ± 0.08* | 0.57 ± 0.11 | 1.21 ± 0.09 | 0.47 ± 0.06 | 2.61 ± 0.21 |
| Supernatant (extracellular) | ||||||
| +/y | — (RP) | 5.4 ± 0.8 | 58.6 ± 4.3 | 45.8 ± 2.9 | 2.0 ± 0.7 | 34.0 ± 0.7 |
| −/y | — (RP) | 5.1 ± 1.7 | 57.5 ± 6.9 | 52.0 ± 5.0 | 2.1 ± 1.15 | 33.7 ± 0.3 |
| +/y | + (Act) | 5.2 ± 1.1 | 50.6 ± 5.8 | 38.3 ± 3.8 | 1.8 ± 0.4 | 33.8 ± 0.6 |
| −/y | + (Act) | 5.4 ± 1.6 | 50.9 ± 4.6 | 47.4 ± 5.4 | 1.9 ± 1.0 | 34.0 ± 0.4 |
| +/y | + (Agg) | 5.1 ± 0.7 | 72.0 ± 4.5 | 44.4 ± 2.9 | 1.2 ± 0.7 | 23.0 ± 6.0 |
| −/y | + (Agg) | 5.1 ± 1.1 | 65.0 ± 5.4 | 50.2 ± 4.2 | 3.6 ± 2.3 | 0 |
Bead-purified platelets (pooled) were incubated with DHE (25 µM), followed by activation (Act; no stirring) or aggregation (Agg; with stirring) with thrombin (0.05 U/mL) and convulxin (50 ng/mL). The oxidation products of DHE, 2-OH-E+, DHE (unreacted probe), E+, E+ dimer, and DHE dimer were quantitated in platelet pellets (intracellular) and supernatant fractions (extracellular). Data are mean ± standard error of the mean (n = 3 or 4 in each group, with 3 technical replicates of each sample).
P < .05 vs. RP for both genotypes.