Columbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo. Aggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study. CBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbβ3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbβ3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation. This study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

中文翻译：

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人血小板活化和体内血管血栓形成的调制通过columbianadin：调节通过整合素α _IIB β ₃内向外的但不是由外而内的信号

Columbianadin（CBN）是从当归中分离出的主要香豆素成分之一。CBN的药理价值已得到充分证明，尤其是在预防多种癌症和止痛活性方面。动脉血栓形成的引人注目的治疗目标是抑制血小板活化，因为血小板活化显着地促成了这些疾病。当前的研究检查了CBN对体外人血小板活化和体内血管血栓形成的影响。这项研究使用了凝集测定法，免疫印迹法，免疫沉淀法，共聚焦显微镜分析法，纤维蛋白凝块收缩法和血栓形成动物。CBN可以显着抑制仅由胶原蛋白刺激的洗涤后人类血小板中的血小板聚集，但对其他激动剂（如凝血酶，花生四烯酸和U46619。CBN明显抑制ATP释放，细胞内（[Ca2 +] i）动员和P-选择素表达。它还抑制磷脂酶C（PLC）γ2，蛋白激酶C（PKC），Akt（蛋白激酶B）和促分裂原活化蛋白激酶（MAPKs；细胞外信号调节激酶[ERK] 1/2和c-胶原激活的血小板中的Jun N末端激酶[JNK] 1/2，但不是p38 MAPK）。腺苷酸环化酶抑制剂SQ22536和鸟苷酸环化酶抑制剂ODQ均未逆转CBN介导的血小板凝集抑制作用。CBN在触发血管扩张剂刺激的磷蛋白磷酸化方面没有显著作用。此外，它通过干扰PAC-1的结合而显着地阻碍了整联蛋白αIIbβ3的活化。但是，它对整合素αIIbβ3介导的由外而内的信号传递没有影响，例如固定化纤维蛋白原上血小板的黏附数目和扩散面积以及凝血酶刺激的纤维蛋白凝块收缩。此外，CBN不会减弱散布在固定化纤维蛋白原上的血小板中FITC-三黄素的结合或蛋白的磷酸化，例如整联蛋白β3，Src和粘着斑激酶。在实验小鼠中，CBN增加了血栓性血小板栓塞形成的阻塞时间。这项研究表明，CBN通过抑制PLCγ2-PKC级联，随后抑制Akt和ERKs / JNKs的活化并影响血小板凝集，对血小板活化表现出优异的活性。所以，