Li Li, Xu Xulin, Lv Keyu, Zheng Guijuan, Wang Hao, Chen Shuai, Huang Lang, Liu Yi, Zhang Yadong, Tang Zhaoming, Zhang Lili, Wang Jinyu, Qiao Jianlin, Li Hongliang, Wang Xuanbin, Yao Guangmin, Fang Chao
Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
Br J Pharmacol. 2023 Feb;180(3):287-307. doi: 10.1111/bph.15964. Epub 2022 Oct 19.
Thrombosis is a major cause of morbidity and mortality worldwide. Platelet activation by exposed collagen through glycoprotein VI (GPVI) and formation of neutrophil extracellular traps (NETs) are critical pathogenic factors for arterial and venous thrombosis. Both events are regulated by spleen tyrosine kinase (Syk)-mediated signalling events. Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown. This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms.
Platelet aggregation was assessed using an aggregometer. Platelet P-selectin exposure, integrin activation and calcium mobilization were determined by flow cytometry. NETs formation was assessed by SYTOX Green staining and immunohistochemistry. Quantitative phosphoproteomics, microscale thermophoresis, in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin. The in vivo effects of asebogenin on arterial thrombosis were investigated using FeCl -induced and laser-induced injury models, whereas those of venous thrombosis were induced by stenosis of the inferior vena cava.
Asebogenin inhibited a series of GPVI-induced platelet responses and suppressed NETs formation induced by proinflammatory stimuli. Mechanistically, asebogenin directly interfered with the phosphorylation of Syk at Tyr525/526, which is important for its activation. Further, asebogenin suppressed arterial thrombosis demonstrated by decreased platelet accumulation and fibrin generation and attenuated venous thrombosis determined by reduced neutrophil accumulation and NETs formation, without increasing bleeding risk.
Asebogenin exhibits potent antithrombotic effects by targeting Syk and is a potential lead compound for the development of efficient and safe antithrombotic agents.
血栓形成是全球发病和死亡的主要原因。暴露的胶原蛋白通过糖蛋白VI（GPVI）激活血小板以及形成中性粒细胞胞外陷阱（NETs）是动脉和静脉血栓形成的关键致病因素。这两个事件均由脾酪氨酸激酶（Syk）介导的信号转导事件调控。紫铆因是一种二氢黄酮，其药理作用在很大程度上尚不清楚。本研究旨在探讨紫铆因的抗血栓作用及其潜在的分子机制。
使用血小板聚集仪评估血小板聚集。通过流式细胞术测定血小板P-选择素暴露、整合素激活和钙动员。通过SYTOX Green染色和免疫组织化学评估NETs形成。进行定量磷酸化蛋白质组学、微量热泳动、体外激酶测定以及分子对接结合动力学模拟以表征紫铆因的靶点。使用FeCl₃诱导和激光诱导损伤模型研究紫铆因对动脉血栓形成的体内作用，而静脉血栓形成的体内作用则通过下腔静脉狭窄诱导。
紫铆因抑制了一系列由GPVI诱导的血小板反应，并抑制了促炎刺激诱导的NETs形成。机制上，紫铆因直接干扰Syk在Tyr525/526位点的磷酸化，这对其激活很重要。此外，紫铆因抑制动脉血栓形成，表现为血小板聚集和纤维蛋白生成减少，并且减轻静脉血栓形成，表现为中性粒细胞聚集和NETs形成减少，而不会增加出血风险。
紫铆因通过靶向Syk发挥强大的抗血栓作用，是开发高效安全抗血栓药物的潜在先导化合物。
