Platelet Research Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health and Innovation Research Institute, Faculty of Health Sciences, Curtin University, Bentley Campus, Office 160, Building 305, Kent Street, Bentley, Perth, WA, 6102, Australia.
Cell Commun Signal. 2018 May 29;16(1):24. doi: 10.1186/s12964-018-0235-0.
The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response.
Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation.
Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y and αIIbβ3 receptors.
In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation.
中性粒细胞细胞外陷阱（NETs）是中性粒细胞从细胞中释放的一种 DNA、组蛋白和中性粒细胞蛋白酶的网状物，最初被证明是宿主防御病原体的一种方式。最近，人们清楚地认识到 NETs 也会在病理条件下释放。在血液中释放的 NETs 可以激活血栓形成并引发血小板反应级联。然而，人们并不清楚这些反应是否是通过直接或间接的相互作用介导的。我们研究了细胞外游离 NETs 是否可以在体外诱导人血小板的聚集，以及 NET 衍生的细胞外 DNA 和组蛋白对血小板激活反应的贡献。
用 PMA 刺激分离的人中性粒细胞产生强烈而一致的 NETs。通过检查 DNA-组蛋白复合物和用 ELISA 定量中性粒细胞弹性蛋白酶来分离和表征细胞外游离 NETs。将 NETs 与洗涤后的人血小板孵育，以评估几种血小板激活反应。使用药理抑制剂，我们探讨了不同的 NET 成分以及主要血小板受体和下游信号通路在 NET 诱导的血小板聚集中的作用。
细胞外游离 NETs 直接诱导剂量依赖性的血小板聚集、致密颗粒分泌和血小板促凝血磷脂酰丝氨酸暴露。令人惊讶的是，我们发现抑制 NET 衍生的 DNA 和组蛋白并不影响 NET 诱导的血小板聚集或激活。我们进一步确定了参与 NET 激活血小板的分子途径。NET 诱导血小板反应的最有效单一调节剂包括 NET 结合的组织蛋白酶 G、血小板 Syk 激酶以及 P2Y 和 αIIbβ3 受体。
体外生成的 NETs 可以直接诱导洗涤后的人血小板明显聚集。用 DNA 酶或肝素预处理 NETs 并不能减少 NET 诱导的人洗涤血小板的激活或聚集。我们进一步确定了血小板对 NET 反应中激活的分子途径。综上所述，我们得出结论，靶向某些血小板激活途径而不是 NET 支架，对 NET 诱导的血小板聚集有更显著的抑制作用。
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