Loss of endogenous Nox2-NADPH oxidase does not prevent age-induced platelet activation and arterial thrombosis in mice

Abstract

Background: Reactive oxygen species are known to contribute to platelet hyperactivation and thrombosis during aging; however, the mechanistic contribution of the specific oxidative pathway remains elusive.

Objectives: We hypothesized that during aging, endogenous Nox2-NADPH oxidase contributes to platelet reactive oxygen species accumulation and that loss of Nox2 will protect from platelet activation and thrombosis.

Methods: We studied littermates of Nox2 knockout (Nox2-KO) and -wild-type (Nox2-WT) mice at young (3-4 months) and old (18-20 months) age. Within platelets, we examined the expression of subunits of NADPH oxidase and enzyme activity, oxidant levels, activation markers, aggregation, and secretion. We also assessed susceptibility to in vivo thrombosis in 2 experimental models.

Results: While aged Nox2-WT mice displayed increased mRNA levels for Nox2, aged Nox2-KO mice showed an increase in Nox4 mRNA. However, neither the protein levels of several subunits nor the activity of NADPH oxidase were found to be altered by age or genotype. Both aged Nox2-WT and aged Nox2-KO mice exhibited similar enhancement in levels of platelet oxidants, granule release, α_IIbβ₃ activation, annexin V binding, aggregation and secretion, and a greater susceptibility to platelet-induced pulmonary thrombosis compared with young mice. In a photochemical injury model, adoptive transfer of platelets from aged Nox2-WT or Nox2-KO mice to the aged host mice resulted in a similar time to develop occlusive thrombus in the carotid artery. These findings suggest that loss of endogenous Nox2 does not protect against age-related platelet activation and arterial thrombosis in mice.

Conclusion: We conclude that Nox2 is not an essential mediator of prothrombotic effects associated with aging.

Keywords: NADPH oxidase; aging; platelets; reactive oxygen species; thrombosis.

Graphical abstract

Figure 1

mRNA for Nox2 is increased in platelets from aged mice. Levels of mRNA were measured in bead-purified platelets using quantitative polymerase chain reaction for catalytic subunits (A) Nox2, (B) Nox4, and (C) Nox1, and regulatory subunits (D) p47Phox and (E) p67Phox of NADPH oxidase and for Nox2-associated subunit (F) p22Phox. Relative expression to young Nox2 wild-type (Nox2-WT) is presented. Data are presented as median with IQR and analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. N = 6 per group. Nox2-KO, Nox2 knockout.

Figure 2

Protein levels of NADPH oxidase subunits or enzymatic activity in platelets are not altered by Nox2 genotype or age. Protein levels were measured in bead-purified platelets by Western blotting for catalytic subunits (A) Nox1 and (B) Nox4 and regulatory subunits (C) p47Phox and (D) p67Phox of NADPH oxidase. The activity of NADPH oxidase was measured in platelet lysate in the presence of cytochrome C and NADPH, and changes in absorbance in the presence or absence of superoxide dismutase were calculated and expressed as the amount of superoxide generated. Data are presented as median with IQR for A and C and as mean ± SE for B, D, and E. Data were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. N = 4 to 8 per group. AKO; aged Nox2 knockout; AW; aged Nox2 wild-type; Nox2-KO, Nox2 knockout; Nox2-WT, Nox2 wild-type; YKO; young Nox2 knockout; YW, young Nox2 wild-type.

Figure 3

Age-induced increase in cellular oxidants in mice is not prevented by Nox2 deficiency. Washed platelets were prepared from young or aged mice deficient in Nox2 (Nox2-KO) or wild-type littermates (Nox2-WT). Levels of intracellular reactive oxygen species detected by oxidation of CM-H2DCF (DCF fluorescence) were measured in platelets at (A) baseline (resting platelets) and upon stimulation with (B) 0.02 U/mL, (C) 0.05 U/mL thrombin, or (D) 0.05 U/mL thrombin ± pegylated (PEG)-catalase. All the samples were analyzed by flow cytometry, and mean fluorescence intensity (MFI) was measured. Data for A–C are presented as MFI (N = 8 to 9 per group). Data for D are presented as fold change from average young Nox2-WT, where MFI obtained with PEG-catalase treatment was subtracted to show a peroxide-dependent increase in DCF fluorescence (N = 5 per group). Data are presented as mean ± SE and analyzed using 2-way anova with Tukey’s analysis for multiple comparisons.

Figure 4

Age-induced increase in cellular and mitochondrial superoxide in mice is not prevented by Nox2 deficiency. Washed platelets were prepared from young or aged mice deficient in Nox2 (Nox2-KO) or wild-type littermates (Nox2-WT). Levels of intracellular superoxide were detected by oxidation of dihydroethidium (DHE) in platelets at (A) baseline (resting platelets) or upon stimulation with (B) 0.05 U/mL thrombin + 50 ng/mL convulxin. The levels of superoxide in platelet mitochondria were detected by MitoSox staining at (C) baseline or upon stimulation with (D) 0.05 U/mL thrombin + 50 ng/mL convulxin. All the samples were analyzed by flow cytometry and mean fluorescence intensity (MFI) was measured. N = 8 to 9 per group. Data are presented as mean ± SE and analyzed using 2-way anova with Tukey’s analysis for multiple comparisons.

Figure 5

Increased platelet activation in aged mice is not mediated through Nox2-NADPH oxidase. Washed platelets were prepared from young or aged mice deficient in Nox2 (Nox2-KO) or wild-type (Nox2-WT) littermates, suspended in Tyrode’s buffer with fluorescent antibody/dye, stimulated with agonists, fixed, and respective fluorescence was measured by flow cytometry. Granule release (P-selectin) and activation of α_IIbβ₃ were measured upon stimulation with (A and C) 0.02 U/mL thrombin or (B and D) 0.05 U/mL thrombin, respectively. Annexin V binding was quantified upon simultaneous activation with (E) 0.05 U/mL thrombin + 50 ng/mL convulxin or (F) 0.1 U/mL thrombin + 100 ng/mL convulxin. Data are presented as mean ± SE and analyzed with 2-way anova with Tukey’s test for multiple comparisons. N = 7 to 9 per group. MFI, mean fluorescence intensity.

Figure 6

Platelets from aged Nox2 wild-type (Nox2-WT) and aged Nox2 knockout (Nox2-KO) mice show similar increases in aggregation and secretion. Washed platelets were prepared from young or aged Nox2-KO and Nox2-WT littermates. (A) Representative tracings for simultaneous aggregation and secretion curves from 4 mice representing each group, where the aggregation curve goes down with an increase in light transmission and the secretion curve goes up with an increase in luminescence. Aggregation curves: black for young Nox2-WT, dark green for young Nox2-KO, dark blue for aged Nox2-WT, and dark red for aged Nox2-KO. Secretion curves: light gray for young Nox2-WT, fluorescent green for young Nox2-KO, light blue for aged Nox2-WT, and orange for aged Nox2-KO mice. (B and C) Quantification of percent aggregation and secretion, respectively, at different time points. Data for B and C are presented as mean ± SE and analyzed using 2-way anova with Tukey’s test for multiple comparisons. N = 7 to 11 per group.

Figure 7

Deficiency of Nox2 does not protect from age-induced increased susceptibility to thrombosis. Susceptibility to (A) pulmonary thrombosis after collagen infusion is shown as a survival curve, and data for the survival curve were analyzed using the log-rank (Mantel–Cox) test. (B) Time to first and stable occlusion in host human interleukin 4 receptor transgenic mice after adoptive transfer of platelets from aged Nox2 wild-type (Nox2-WT) or aged Nox2 knockout (Nox2-KO) mice. ∗∗P < .01, where the dotted black line indicates a difference between young and aged Nox2-KO, and the solid black line indicates a difference between young and aged Nox2-WT mice. Data for B are presented as mean ± SE and analyzed with an unpaired t-test. N = 6 to 10 per group.