C2238 ANP gene variant promotes increased platelet aggregation through the activation of Nox2 and the reduction of cAMP

Abstract

Subjects carrying the C2238 variant of the atrial natriuretic peptide (ANP) gene have a higher occurrence of stroke and acute coronary syndrome, suggesting an increased predisposition to acute thrombotic events in these subjects. We evaluated for the first time the direct effects of mutant ANP (C2238/αANP) on platelet activation in vitro and in human subjects. In vitro, platelets were incubated with no peptide, with T2238/αANP (WT) or with C2238/αANP at different concentrations. C2238/αANP (10-10 M) induced higher collagen-induced platelet aggregation with respect to both control without ANP and T2238/αANP. This effect was even stronger at a higher concentration (10-6 M). Mechanistically, C2238/αANP significantly lowered platelet cAMP levels, increased ROS production and activated Nox2, with respect to both control and T2238/αANP. Forskolin, a cAMP activator, and sNOX2-tat, a Nox2 inhibitor, significantly reduced the pro-aggregant effects of C2238/αANP. In vivo, we found that platelet aggregation resulted to be higher in patients with atrial fibrillation carrying the C2238 ANP gene variant with respect to non-carriers. In conclusions, C2238/αANP promotes platelet aggregation through the activation of Nox2 and the reduction of cAMP.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

C2238/αANP induces platelet activation. (A,B) Platelets were incubated without the peptide or with either T2238/αANP or C2238/αANP at the specified concentration (10−6 M or 10−10 M) and the platelet activation was triggered by a sub-threshold concentration of collagen (0.3 µg/ml). Platelet aggregation (A) and the concentration of the soluble CD40 ligand released by platelets (B) were assessed. N = 12. The results were expressed as mean ± SEM. In all the groups, a similar volume of solvent was added.

Figure 2

C2238/αANP reduces cAMP levels and promotes platelet oxidative stress. (A–C) Platelets were incubated without the peptide or with either T2238/αANP or C2238/αANP at the specified concentration (10−6 M or 10−10 M). Forskolin (10 µM) was also used where specified. After incubation and platelet stimulation with a sub-threshold concentration of collagen (0.3 µg/ml), intracellular cAMP levels (A), platelet hydrogen peroxide production (B) and platelet Nox2 activation (C) were assessed. N = 12. The results were expressed as mean ± SEM. In all the groups, a similar volume of solvent was added.

Figure 3

C2238/αANP induces platelet aggregation and oxidative stress by the inhibition of cAMP levels and activation of Nox2. (A–D) Platelets were incubated without the peptide or with C2238/αANP at the specified concentration (10−6 M or 10−10 M). Forskolin (10 µM) or sNOX2-tat (50 µM) were also used where specified. After incubation and platelet stimulation with a sub-threshold concentration of collagen (0.3 µg/ml), platelet aggregation (A), soluble CD40 ligand release (B), platelet hydrogen peroxide levels (C) and platelet Nox2 activity were assessed. N = 12. The results were expressed as mean ± SEM. In all the groups, a similar volume of solvent was added.

Figure 4

C2238 ANP gene variant is associated with increased platelet aggregation and oxidative stress. (A–D) Platelet aggregation (A), soluble CD40 ligand release (B), platelet hydrogen peroxide levels (C) and platelet Nox2 activity were assessed in subjects not carrying the variant and in subjects carrying the C2238 ANP gene variant. The results were expressed as mean ± SEM.