Abstract
Background: High levels of proprotein convertase subtilisin/kexin 9 (PCSK9) is predictive of cardiovascular events (CVEs) in atrial fibrillation (AF). We hypothesized that PCSK9 may directly induce platelet activation (PA). Methods: We measured platelet aggregation, recruitment, Thromboxane B2 (TxB2) formation and soluble P-selectin levels as markers of PA and soluble Nox2-derived peptide (sNox2-dp), H2O2, isoprostanes and oxidized Low-Density-Lipoprotein (oxLDL) to analyze oxidative stress (OS) in 88 patients having PCSK9 values < (n = 44) or > (n = 44) 1.2 ng/mL, balanced for age, sex and cardiovascular risk factors. Furthermore, we investigated if normal (n = 5) platelets incubated with PCSK9 (1.0–2.0 ng/mL) alone or with LDL (50 µg/mL) displayed changes of PA, OS and down-stream signaling. Results: PA and OS markers were significantly higher in patients with PCSK9 levels > 1.2 ng/mL compared to those with values < 1.2 ng/mL (p < 0.001). Levels of PCSK9 significantly correlated with markers of PA and OS. Platelets incubation with PCSK9 increased PA, OS and p38, p47 and Phospholipase A2 (PLA2) phosphorylation. These changes were amplified by adding LDL and blunted by CD36 or Nox2 inhibitors. Co-immunoprecipitation analysis revealed an immune complex of PCSK9 with CD36. Conclusions: We provide the first evidence that PCSK9, at concentration found in the circulation of AF patients, directly interacts with platelets via CD36 receptor and activating Nox2: this effect is amplified in presence of LDL.
Highlights
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is principally present in the liver, where it is able to modulate Low-Density-Lipoprotein (LDL) cholesterol uptake acting on its LDL receptor (LDL-R).The gain of function variants of proprotein convertase subtilisin/kexin 9 (PCSK9) gene is associated with increased levels of circulating LDL cholesterol, and a higher risk of cardiovascular events (CVE) [1]
(C) and serum Thromboxane B2 (TxB2) (D) in 44 atrial fibrillation (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)
To analyse the pathway involved in PCSK9-dependent platelet activation by reactive oxygen species (ROS), we studied p47phox translocation on the platelet membrane, the role of p38MAP kinase, which is implicated in p47phox activation [16] as final and key event upstream of Nox2 activation, and cPLA2 phosphorylation, a key enzyme for generation of eicosanoids
Summary
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is principally present in the liver, where it is able to modulate Low-Density-Lipoprotein (LDL) cholesterol uptake acting on its LDL receptor (LDL-R). A recent prospective study investigated PCSK9 levels in atrial fibrillation (AF) patients and revealed that high plasma levels of PCSK9 were significantly associated with urinary 11-dehydro-Thromboxane. PCSK9 was associated with increased platelet aggregation in patients with acute coronary syndrome [5] and treatment with anti-PCSK9 mAb was found to modulate platelet reactivity [6]. Among the mechanisms involved in PA, ROS and eventually oxidized LDL (ox-LDL) play an important role via triggering the intra-cellular signaling of platelet activation [9]. To substantiate that PCSK9 could be biologically active on platelet activation, we performed in vitro studies to assess (i) the interplay between PCSK9 and platelet activation, and (ii) oxidative stress, and (iii) to investigate the molecular mechanism involved
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