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Redox-dependent regulation of platelets
Role of NOX1 and NOX2 in mediating the effect on platelets of weak agonists/positive modulators oxidized LDL (oxLDL) and amyloid beta (Aβ1-42)
Using these tools (NoxA1ds26 and Nox2ds-tat27,28), we determined that NOX1 or NOX2 play an equivalent role in the generation of superoxide anion in response to oxLDL (Figure 6A) and that both enzymes are required for the stimulation of the modest aggregation induced by this modulator (Figure 6B). As suggested by the inhibition of aggregation by PEG-SOD, O2•− is the oxidant molecule required for the activation of platelets by oxLDL. It is important to note how oxLDL elicits a very modest aggre- gation (both as size and kinetic of the response), which con- forms to the designation of oxLDL as a modulator rather
AB
than agonist (i.e. exerting its effect by enhancing the responsiveness of platelets to low agonist levels). In fact, low concentrations of collagen (Figure 6C) or thrombin (Figure 6D) characterized by the ability to induce no/low aggregation, resulted able to induce a robust platelet aggre- gation in the presence of ox-LDL. The synergistic effect of oxLDL in combination with thrombin or collagen was inhibited by NoxA1ds (Figure 6C) or Nox2ds-tat (Figure 6D), respectively. We could not test NoxA1ds on ox-LDL + collagen or Nox2ds-tat on oxLDL + thrombin, because, as shown above, NoxA1ds inhibits collagen directly and Nox2ds-tat inhibits thrombin directly. Similarly, Aβ1-42 induces O2•− formation via activation of NOX1 and NOX2 (Figure 7A), which leads to a modest platelet aggregation
C
D
Figure 2. NOX1 is specifically involved in platelet activation by collagen but not thrombin. 1-hydroxy-3-methoxycarbonyl- 2,2,5,5-tetramethylpyrrolidine (CMH) was utilized for the detection of oxygen radi- cals generated by platelets. 10 μg/mL col- lagen (A) or 0.1 unit/mL thrombin (B) were tested. 10 μM of NoxA1ds abolished the electron paramagnetic resonance (EPR) response measured in the pres- ence of collagen. The scrambled peptide at the same concentration (scNoxA1ds) was used as a negative control. Interestingly, the inhibition of NOX1 by NoxA1ds also inhibited collagen-depen- dent (C), but not thrombin-dependent (D) platelet aggregation. Aggregation curves for up to 5 minutes (min) are shown, while EPR resonance readings were taken after 10 min of stimulation. Examples of EPR traces and aggregation curves are representative of 3 or more independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test for EPR. *P<0.05 compared to resting platelets or t-test for aggregation. *P<0.05 compared to scrambled control. N=3 for (A-D).
haematologica | 2019; 104(9)
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