Abstract：
OBJECTIVE: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties.
METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA.
RESULTS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole.
CONCLUSIONS: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.
METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA.
RESULTS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole.
CONCLUSIONS: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.
摘要：
目的：在许多细菌和病毒引起的疾病中，血小板聚集失调是一种致命的疾病。然而,经典的抗血栓药物不能完全预防免疫血栓形成，由于未解决的炎症机制。因此，靶向血小板过度活化和炎症可能为疾病提供新的治疗选择，以免疫血栓形成为特征，如COVID-19和脓毒症。这项研究的目的是研究1.8-桉树脑的抗聚集作用和作用方式，从桉树叶的精油中提取的单萜，以其抗炎特性而闻名。
方法：通过测量血小板活化标志物的表达和释放来监测血小板活性，即，P-选择素,CD63和CCL5，以及血小板聚集，用1.8-桉树脑治疗并用几种经典刺激和细菌刺激。激酶活性测定用于阐明作用模式，然后通过Westernblot和ELISA详细分析腺苷酸环化酶（AC）-环磷酸腺苷（cAMP）-蛋白激酶A（PKA）途径的参与。
结果：1.8-桉树脑阻止血小板活化标志物的表达和释放，以及血小板聚集，在用经典刺激和免疫激动剂诱导聚集后。机械上,1.8-桉树脑影响AC-cAMP-PKA通路的激活，导致更高的cAMP水平和血管扩张剂刺激的磷蛋白（VASP）磷酸化。最后,阻断腺苷A2A受体逆转了1.8-桉树脑的抗血栓作用。
结论：鉴于公认的1.8-桉树脑的抗炎特性，再加上我们的发现，1.8-桉树脑可能成为治疗以血小板活化和异常炎症为标志的疾病的有希望的候选者。
方法：通过测量血小板活化标志物的表达和释放来监测血小板活性，即，P-选择素,CD63和CCL5，以及血小板聚集，用1.8-桉树脑治疗并用几种经典刺激和细菌刺激。激酶活性测定用于阐明作用模式，然后通过Westernblot和ELISA详细分析腺苷酸环化酶（AC）-环磷酸腺苷（cAMP）-蛋白激酶A（PKA）途径的参与。
结果：1.8-桉树脑阻止血小板活化标志物的表达和释放，以及血小板聚集，在用经典刺激和免疫激动剂诱导聚集后。机械上,1.8-桉树脑影响AC-cAMP-PKA通路的激活，导致更高的cAMP水平和血管扩张剂刺激的磷蛋白（VASP）磷酸化。最后,阻断腺苷A2A受体逆转了1.8-桉树脑的抗血栓作用。
结论：鉴于公认的1.8-桉树脑的抗炎特性，再加上我们的发现，1.8-桉树脑可能成为治疗以血小板活化和异常炎症为标志的疾病的有希望的候选者。
