Cao Hang, Umbach Anja T, Bissinger Rosi, Gawaz Meinrad, Lang Florian
Department of Internal Medicine III, Tübingen, Germany.
Department of Physiology, Eberhard-Karls-University, Tübingen, Germany.
Cell Physiol Biochem. 2018;45(4):1707-1716. doi: 10.1159/000487778. Epub 2018 Feb 23.
BACKGROUND/AIMS: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis.
Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 - 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE.
In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib.
The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.
背景/目的：间变性淋巴瘤（酪氨酸）激酶（ALK）抑制剂色瑞替尼可触发肿瘤细胞凋亡和红细胞的 eryptosis。血小板可能同样进入类似凋亡的状态，这可能由胶原相关肽（CRP）激活引发。CRP 诱导的血小板凋亡的特征是细胞膜紊乱，磷脂酰丝氨酸暴露于血小板表面以及细胞收缩，之前伴有 Ca2+通道 Orai1 的外化、胞质 Ca2+活性（[Ca2+]i）增加、活性氧（ROS）形成以及半胱天冬酶激活。本研究探讨了色瑞替尼是否触发血小板凋亡和/或改变 CRP 诱导的凋亡。
从野生型小鼠分离的血小板在无 CRP（2 μg/ml 或 5 μg/ml）预处理或有 2.5 - 15 分钟 CRP 预处理的情况下，暴露于色瑞替尼（1.5 μg/ml）30 分钟。采用流式细胞术从 Fluo-3 荧光估计胞质 Ca2+活性（[Ca2+]i），从 2',7'-二氯二氢荧光素二乙酸酯荧光估计 ROS 丰度，从 P-选择素丰度估计血小板脱颗粒，从 αIIbβ3 整合素丰度估计整合素激活，使用活性半胱天冬酶-3 染色试剂盒估计半胱天冬酶活性，从膜联蛋白-V 结合估计磷脂酰丝氨酸丰度，从前向散射估计血小板体积，并使用 CD9-APC 和 CD9-PE 染色估计聚集。
在没有 CRP 的情况下，色瑞替尼轻微但显著降低了[Ca2+]i，而没有显著改变其他测量参数。CRP 显著增加了[Ca2+]i、ROS 丰度、P-选择素丰度、活化的 αIIbβ3 整合素、膜联蛋白-V 结合、半胱天冬酶活性以及聚集，并降低了细胞体积，在有色瑞替尼存在的情况下，所有这些效应均显著减弱。
本观察结果揭示了色瑞替尼一种新的、意想不到的作用，即抑制 CRP 诱导的血小板激活和凋亡。
