PCSK9 Regulates Nox2-Mediated Platelet Activation via CD36 Receptor in Patients with Atrial Fibrillation

Figures

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  Figure 1. Proprotein convertase subtilisin/kexin 9PCSK9 and platelet activation in atrial fibrillation (AF) patients. Platelet aggregation (A), platelet recruitment (B), plasma soluble P-selectin (sP-selectin) (C) and serum TxB2 (D) in 44 AF patients with plasma levels of PCSK9 < 1.2 ng/mL and 44 AF patients with plasma levels of PCSK9 > 1.2 ng/mL. (Data are represented as median and IQR. ** p < 0.001).

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  Figure 2. PCSK9 and oxidative stress in AF patients. Nox2 activation (A), H2O2 production (B), ox-LDL formation (C) and 8-iso-PGF2α (D) biosynthesis in 44 AF patients with plasma levels of PCSK9 < 1.2 ng/mL and 44 AF patients with plasma levels of PCSK9 > 1.2 ng/mL. (Data are represented as median and IQR. **p < 0.001).

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  Figure 3. PCSK9 and platelet activation. Platelet aggregation (A) with relative representative tracing (B) and platelet TxB2 (C) evaluated in platelet rich plasma (PRP) treated with PCSK9 (1 and 2 ng/mL) and stimulated with subthreshold concentration (STC) of collagen (0.25 μg/mL) in presence or not of anti-CD36, anti-LOX1, Nox2ds-tat and anti-PCSK9. (n = 5) (*p < 0.05, **p < 0.001 for paired analyses).

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  Figure 4. PCSK9 and oxidative stress. Nox2 activation (A), H2O2 production (B), 8-iso-PGF2α-III (C) and ox-LDL formation (D) evaluated in PRP treated with PCSK9 (1 and 2 ng/mL) and stimulated with STC of collagen (0.25 μg/mL) in presence or less of anti-CD36, anti-LOX1, Nox2ds-tat and anti-PCSK9. (n = 5) (**p < 0.001 for paired analyses).

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  Figure 5. Intra-signalling pathway of platelet activation PCSK9-mediated: p38MAP Kinase, p47phox and PLA2 phosphorylation. p38 phosphorylation, p47phox phosphorylation and cPLA2 phosphorylation was analysed in platelets incubated with PCSK9 (1–2 ng/mL) and stimulated with subthreshold concentration (STC) of collagen (0.25 µg/mL) (A–C) in presence or less of anti-CD36, anti-LOX1, anti-PCSK9 (A–C) and incubated with or without SB (A), Nox2ds-tat (B) and AACOCF3 (C) (n = 3 independent experiments; *p < 0.05 **p < 0.001 in quantitative analysis). A representative Western blot of p38, p47phox and cPLA2 phosphorylation (B–C).

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  Figure 6. PCSK9 and platelet activation LDL-mediated. Platelet aggregation (A) with relative representative tracing (B), TxB2 production (C) and conjugated dienes formation (D) evaluated in washed platelet treated with PCSK9 alone (1–2 ng/mL) and stimulated with subthreshold concentration (STC) of collagen (0.25 µg/mL) in presence or less of exogenous LDL (50 µg/mL), Nox2ds-tat and anti-CD36. (n = 5; *p < 0.05, **p < 0.001 for paired analyses).

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  Figure 7. PCSK9 activates platelets by CD36 signalling. Densitometric analysis of Western blotting (WB) showing coimmunoprecipitation of CD36 and LOX1 with PCSK9 in human platelets stimulated with PCSK9 2 ng/mL (A). A representative Western blot bands of immune complexes CD36-PCSK9 and LOX1-PCSK9, the lane with 10% of the extract loaded before of IP (input) are shown (B). Results are representative of three independent experiments.