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D. Vara et al.
ly suggest that platelet O2•− acts intracellularly in these responses, as only cell permeable scavenger can affect their activity (i.e. PEG-SOD), while hydrogen peroxide is likely to be formed by dismutation of intracellular O2•− and released extracellularly, where it potentiates platelet responses induced by thrombin (as proved by the effect of non-cell permeable catalase shown in Online Supplementary
Figure S6). These findings add significantly to our under- standing of platelet redox regulation and are in agreement with previous suggestions of an intracellular function for O2•− .32,33 It is also in agreement with the observation of a positive regulatory role for extracellular hydrogen peroxide in thrombin-induced responses34 and a role for extracellular oxidants in the regulation of platelet surface receptor func-
AB
C
D
Figure 7. NOX1 and NOX2 are required for the induction of superoxide anion formation and platelet activation by amyloid peptide β 1-42. Oxygen radical generation (A) and platelet aggregation (B) in response to amyloid peptide β 1-42 (Aβ1-42) were measured as described. 10 μM of scrambled NoxA1ds (scNoxA1ds), NoxA1ds, scrambled Nox2ds-tat (scNox2ds-tat), Nox2ds-tat or 100 unit/mL of PEG-SOD were pre-incubated 10 minutes (min) before platelet stimulation with 20 μM Aβ1-42. 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 (A and B). The effect of Aβ1-42 as platelet modulator was investigated by traditional aggre- gometry (C and D). Pre-incubation with 20 μM Aβ1-42 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 Aβ1-42 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 Aβ1-42). Aggregation curves are representative of 4 independent experiments.
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