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D. Vara et al.
(Figure 7B). On the other hand, similarly to oxLDL, Aβ1-42 displays the ability to synergistically increase the aggrega- tion response to low concentrations of collagen (Figure 7C) or thrombin (Figure 7D). As proved with inhibitory pep- tides NoxA1ds26 and Nox2ds-tat,27,28 the synergistic effect on collagen- or thrombin-induced aggregation is NOX1- or NOX2-dependent, respectively. We could not test NoxA1ds on Aβ1-42 + collagen or Nox2ds-tat on Aβ1-42 + thrombin, because, as shown above, NoxA1ds inhibits collagen direct- ly and Nox2ds-tat inhibits thrombin directly. Transgenic mice NOX1-/- and NOX2-/- were utilized to assess the NOX- dependence of the responses to oxLDL and Aβ1-42. As ox- LDL coating of surfaces is not commonly used and there is no accepted protocol for this procedure, we tested the
AB
effect of oxLDL added to mouse blood on thrombus forma- tion on low levels (i.e. 0.05 mg/mL) of collagen coating (Figure 8A). These data showed that oxLDL potentiates the thrombus formation on collagen in a NOX2-dependent manner (as it was not evident in NOX2-/- blood). NOX1 ablation inhibits collagen responses directly, therefore we could not investigate the role of NOX1 in the potentiation of responses to this agonist by oxLDL. The ablation of either NOX1 or NOX2 silencing significantly also impairs thrombus formation at physiological arterial shear on absorbed Aβ1-42 (1,000 sec-1) (Figure 8C). At low shear (200 sec-1), mouse platelets display low levels of adhesion to absorbed Aβ1-42 without formation of thrombi, which was inhibited by NOX1 genetic silencing but unaffected by
CD
Figure 3. NOX2 is activated by both collagen and thrombin, but essential only for platelet aggre- gation induced by thrombin. 1- hydroxy-3-methoxycarbonyl- 2,2,5,5-tetramethylpyrrolidine (CMH) was utilized for the detec- tion of oxygen radicals generated by platelets. 10 μg/mL collagen (A) or 0.1 unit/mL thrombin (B) were tested. 10 μM of Nox2ds-tat inhibited the electron paramag- netic resonance (EPR) response measured in the presence of either collagen or thrombin, although the collagen-dependent response remained significantly higher than resting levels of oxy- gen radical formation. The scram- bled peptide at the same concen- tration (scNox2ds-tat) was used as a negative control. Interestingly, the inhibition of NOX2 by Nox2ds-tat also inhibit- ed thrombin-dependent (D), but not collagen-dependent (C) platelet aggregation. Examples of EPR traces and aggregation curves are representative of 3 or more independent experiments. Statistical analysis was per- formed 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=4 for (B and D), n=5 for (A), and n=7 for (C).
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haematologica | 2019; 104(9)