Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450018, China.
Biochem Pharmacol. 2021 Jan;183:114315. doi: 10.1016/j.bcp.2020.114315. Epub 2020 Nov 3.
Previously, we discovered that the activation of nucleotide-binding oligomerization domain 2 (NOD2) enhances platelet activation. We here investigated the antiplatelet and antithrombotic potential of GSK669, a NOD2 antagonist.
Effects of GSK669 on platelet functions, reactive oxygen species (ROS) and proinflammatory cytokine generation were detected. NOD2-/- platelets were used to confirm GSK669 target. The interaction between GSK669 and glycoprotein VI (GPVI) was detected using surface plasmon resonance (SPR) spectroscopy. GPVI downstream signaling was examined by Western blot. The antithrombotic and antioxidative effects were investigated using mouse mesenteric arteriole thrombosis model and pulmonary embolism model.
GSK669 significantly inhibits platelet proinflammatory cytokine release induced by muramyl dipeptide, platelet aggregation, ATP release, and ROS generation induced by collagen and collagen related peptide (CRP). Platelet spreading and clot retraction are also inhibited. GSK669 also decreases collagen-induced phosphorylation of Src, Syk, PLCγ2, and Akt. The antiplatelet effect of GSK669 is NOD2-independent and mediated by GPVI antagonism. Consistent with its antiplatelet activity as a GPVI antagonist, GSK669 inhibits platelet adhesion on collagen in flow condition. Notably, GSK669 inhibits mouse mesenteric arteriole thrombosis similarly to aspirin without bleeding. The antithrombotic effect of GSK669 is further confirmed in the pulmonary embolism model; decreased malonaldehyde (MDA) and increased superoxide dismutase (SOD) levels in mouse plasma reveal a significant antioxidant effect of GSK669.
Beyond its anti-inflammatory effect as a NOD2 antagonist, GSK669 is also an efficient and safe antiplatelet agent combined with antioxidant effect by targeting GPVI. An antiplatelet agent bearing antioxidative and anti-inflammatory activities without bleeding risk may have therapeutic advantage over current antiplatelet drugs for atherothrombosis.
此前，我们发现核苷酸结合寡聚化结构域 2（NOD2）的激活可增强血小板激活。我们在此研究了 NOD2 拮抗剂 GSK669 的抗血小板和抗血栓形成作用。
检测 GSK669 对血小板功能、活性氧（ROS）和促炎细胞因子生成的影响。使用 NOD2-/-血小板来确认 GSK669 的作用靶点。使用表面等离子体共振（SPR）光谱检测 GSK669 与糖蛋白 VI（GPVI）的相互作用。通过 Western blot 检测 GPVI 下游信号转导。使用小鼠肠系膜小动脉血栓形成模型和肺栓塞模型研究抗血栓形成和抗氧化作用。
GSK669 可显著抑制 muramyl dipeptide 诱导的血小板促炎细胞因子释放、胶原和胶原相关肽（CRP）诱导的血小板聚集、ATP 释放和 ROS 生成。血小板铺展和凝块回缩也受到抑制。GSK669 还可降低胶原诱导的Src、Syk、PLCγ2 和 Akt 磷酸化。GSK669 的抗血小板作用与 NOD2 无关，而是通过拮抗 GPVI 介导的。与作为 GPVI 拮抗剂的抗血小板作用一致，GSK669 可抑制胶原在流态下诱导的血小板黏附。值得注意的是，GSK669 对阿司匹林的抑制小鼠肠系膜小动脉血栓形成作用相似，且无出血。GSK669 在肺栓塞模型中的抗血栓形成作用进一步得到证实；小鼠血浆中丙二醛（MDA）水平降低和超氧化物歧化酶（SOD）水平升高表明 GSK669 具有显著的抗氧化作用。
除了作为 NOD2 拮抗剂的抗炎作用外，GSK669 还是一种有效的、安全的抗血小板药物，通过靶向 GPVI 兼具抗氧化作用。具有抗氧化和抗炎作用且无出血风险的抗血小板药物可能比目前用于动脉血栓形成的抗血小板药物具有治疗优势。
