Dyslipidemia-associated atherogenic oxidized lipids induce platelet hyperactivity through phospholipase Cγ2-dependent reactive oxygen species generation

Oxidized low-density lipoprotein (oxLDL) and associated oxidized phosphocholine-headgroup phospholipids (oxPCs) activate blood platelets through ligation of the scavenger receptor CD36. Previously, we found that oxLDL stimulated phosphorylation of phospholipase Cγ2 (PLCγ2). However, the functional relevance of PLCγ2 phosphorylation in oxLDL-mediated platelet hyperactivity remained elusive. Here, we set out to explore the functional importance of PLCγ2 in oxLDL-mediated platelet activation using human and genetically modified murine platelets. The CD36-specific oxidized phospholipid (oxPC_CD36) triggered the generation of reactive oxygen species (ROS) in platelets under static and arterial flow conditions. The ROS generation in response to oxPC_CD36 was sustained for up to 3 h but ablated in CD36- and PLCγ2-deficient platelets. The functional importance of ROS generation in response to atherogenic lipid stress was examined through measurement of P-selectin expression. OxPC_CD36 induced P-selectin expression, but required up to 60 min incubation, consistent with the timeline for ROS generation. P-selectin expression was not observed in CD36- and PLCγ2-deficient mice. The ability of oxPC_CD36 and oxLDL to stimulate P-selectin expression was prevented by incubation of platelets with the ROS scavenger N-acetyl-cysteine (NAC) and the NOX-2 inhibitor gp91ds-tat, but not with the NOX-1 inhibitor ML171. In summary, we provide evidence that prolonged exposure to oxLDL-associated oxidized phospholipids induces platelet activation via NOX-2-mediated ROS production in a CD36- and PLCγ2-dependent manner.

氧化型低密度脂蛋白（oxLDL）及其结合的带有氧化磷脂酰胆碱头部基团的氧化磷脂（oxPCs）可通过结合清道夫受体CD36（scavenger receptor CD36）激活血液血小板。此前本团队发现，oxLDL可刺激磷脂酶Cγ2（PLCγ2）发生磷酸化，但PLCγ2磷酸化在oxLDL介导的血小板过度活化中的功能意义仍未明确。本研究旨在借助人源血小板与基因修饰的小鼠血小板，探究PLCγ2在oxLDL介导的血小板活化过程中的功能重要性。CD36特异性氧化磷脂（oxPC_CD36）可在静态及动脉血流条件下诱导血小板产生活性氧（ROS）。oxPC_CD36诱导的ROS生成可持续长达3小时，但在CD36缺陷型与PLCγ2缺陷型血小板中该反应完全被阻断。本研究通过检测P选择素表达，探究了ROS生成在致动脉粥样硬化脂质应激中的功能重要性。oxPC_CD36可诱导P选择素表达，但该过程需要长达60分钟的孵育，与ROS生成的时间进程相符。在CD36缺陷型与PLCγ2缺陷型小鼠中未检测到P选择素表达。若将血小板与活性氧清除剂N-乙酰半胱氨酸（NAC）及NOX-2抑制剂gp91ds-tat共同孵育，可抑制oxPC_CD36与oxLDL诱导P选择素表达的能力；而使用NOX-1抑制剂ML171则无此抑制效果。综上，本研究证实，长期暴露于oxLDL结合的氧化磷脂，可通过依赖CD36与PLCγ2的途径，经由NOX-2介导的ROS生成诱导血小板活化。