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Redox-dependent regulation of platelets
apoptosis),30 this study for the first time highlights a signifi- cant difference in the role of hydrogen peroxide in the responses to collagen and thrombin. Our observations on the link between thrombin-dependent activation and hydrogen peroxide contradict previous reports pointing to a role for hydrogen peroxide in collagen but not thrombin signaling.31 The experimental differences in our and previ-
A
ous studies are extensive and the poor specificity of the tools used for older studies (e.g. 2',7'–dichlorofluorescein diacetate or DCFDA) are potentially responsible for these discrepancies.
Another important addition to our understanding of redox regulation of platelets is the clarification of whether oxidants act intracellularly or extracellularly. Our data clear-
B
C
D
Figure 6. NOX1 and NOX2 are required for the induction of superoxide anion formation and platelets activation by oxidized low density lipoprotein (oxLDL). Oxygen radical generation (A) and platelet aggregation (B) in response to oxLDL were measured as described. 10 μM of scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scram- bled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat or 100 unit/mL of PEG-SOD were pre-incubated 10 minutes (min) before platelet stimulation with 50 ng/mL oxLDL. Examples of electron paramagnetic resonance (EPR) traces and aggregation curves are representative of 4 independent experiments. Statistical analysis was per- formed by one-way ANOVA with Bonferroni post-hoc test. *P<0.05. N=3 for (B) and n=4 for (A). The effect of oxLDL as platelet modulator was investigated by tradi- tional aggregometry (C and D). Pre-incubation with 50 ng/mL oxLDL for 10 min was followed by stimulation with 3 μg/mL collagen (C) or 0.03 unit/mL thrombin (D). In order to test the dependence of the modulatory effect of oxLDL on NOX1 and NOX2, 10 μM scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat were pre-incubated (10 min before addition of oxLDL). Aggregation curves are representative of 4 independent experiments.
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