Cammisotto Vittoria, Pastori Daniele, Nocella Cristina, Bartimoccia Simona, Castellani Valentina, Marchese Cinzia, Scavalli Antonio Sili, Ettorre Evaristo, Viceconte Nicola, Violi Francesco, Pignatelli Pasquale, Carnevale Roberto
Department of General Surgery and Surgical Speciality Paride Stefanini, Sapienza University of Rome, 00161 Rome, Italy.
Department of Clinical, Internal Medicine and Cardiovascular Sciences, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy.
Antioxidants (Basel). 2020 Apr 2;9(4):296. doi: 10.3390/antiox9040296.
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).
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), HO, isoprostanes and oxidized Low-Density-Lipoprotein (oxLDL) to analyze oxidative stress (OS) in 88 patients having PCSK9 values < ( = 44) or > ( = 44) 1.2 ng/mL, balanced for age, sex and cardiovascular risk factors. Furthermore, we investigated if normal ( = 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.
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 ( < 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.
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.
高水平的前蛋白转化酶枯草溶菌素/kexin 9（PCSK9）可预测心房颤动（AF）患者的心血管事件（CVE）。我们推测PCSK9可能直接诱导血小板活化（PA）。
我们测量了血小板聚集、募集、血栓素B2（TxB2）形成和可溶性P-选择素水平作为PA的标志物，以及可溶性Nox2衍生肽（sNox2-dp）、血红素加氧酶（HO）、异前列腺素和氧化型低密度脂蛋白（oxLDL），以分析88例PCSK9值<（=44）或>（=44）1.2 ng/mL患者的氧化应激（OS），这些患者在年龄、性别和心血管危险因素方面保持平衡。此外，我们研究了单独用PCSK9（1.0 - 2.0 ng/mL）或与低密度脂蛋白（LDL，50 µg/mL）一起孵育的正常（=5）血小板是否表现出PA、OS和下游信号传导的变化。
与PCSK9水平<1.2 ng/mL的患者相比，PCSK9水平>1.2 ng/mL的患者的PA和OS标志物显著更高（<0.001）。PCSK9水平与PA和OS标志物显著相关。用PCSK9孵育血小板会增加PA、OS以及p38、p47和磷脂酶A2（PLA2）的磷酸化。添加LDL会放大这些变化，而CD36或Nox2抑制剂会使其减弱。免疫共沉淀分析揭示了PCSK9与CD36的免疫复合物。
我们提供了首个证据，即在AF患者循环中发现的浓度下，PCSK9通过CD36受体与血小板直接相互作用并激活Nox2：在存在LDL的情况下，这种作用会放大。
