Cao Hang, Bhuyan Abdulla Al Mamun, Umbach Anja T, Bissinger Rosi, Gawaz Meinrad, Lang Florian
Department of Internal Medicine III, Tuebingen, Germany.
Department of Physiology I, Eberhard-Karls-University, Tuebingen, Germany.
Cell Physiol Biochem. 2017;43(6):2264-2276. doi: 10.1159/000484377. Epub 2017 Oct 27.
BACKGROUND/AIMS: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP).
Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 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 thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbβ3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib.
Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.
背景/目的：表皮生长因子受体（EGFR）酪氨酸激酶抑制剂阿法替尼用于治疗多种恶性肿瘤。阿法替尼通过触发肿瘤细胞凋亡至少部分有效。血小板可能同样会发生凋亡，其特征为半胱天冬酶3激活、细胞皱缩和磷脂酰丝氨酸易位。然而，阿法替尼对血小板的作用从未被报道过。本研究探讨了在不存在和存在血小板激活剂凝血酶或胶原相关肽（CRP）的情况下，用阿法替尼处理血小板是否会改变血小板激活和凋亡。
将从野生型小鼠分离的血小板暴露于阿法替尼（18 µg/ml）30分钟，不进行后续处理或随后用凝血酶（0.005 U/ml或0.01 U/ml）或CRP（2 µg/ml或5 µg/ml）处理。采用流式细胞术，用特异性抗体估计血小板表面Orai1丰度，通过Fluo-3荧光估计胞质Ca2+活性（[Ca2+]i），通过P-选择素丰度估计血小板脱颗粒，通过αIIbβ3整合素丰度估计整合素激活，使用活性半胱天冬酶-3染色试剂盒检测半胱天冬酶活性，通过膜联蛋白-V结合估计磷脂酰丝氨酸丰度，通过前向散射估计血小板体积，并使用CD9-APC和CD9-PE染色检测聚集情况。
在不存在凝血酶和CRP的情况下，给予阿法替尼（18 µg/ml）会轻微但显著增加[Ca2+]i和膜联蛋白-V结合，但不会显著改变Orai1丰度、P-选择素丰度、活化的αIIbβ3整合素、细胞体积、半胱天冬酶活性和聚集。血小板暴露于0.005 U/ml或0.01 U/ml凝血酶或2 µg/ml或5 µg/ml CRP后，Orai1丰度、[Ca2+]i、P-选择素丰度、αIIbβ3整合素活性、膜联蛋白-V结合、半胱天冬酶活性和聚集均显著增加，同时前向散射显著降低，所有这些效应均被阿法替尼显著减弱（凝血酶）或几乎消除（CRP）。
阿法替尼是血小板激活、血小板凋亡和血小板聚集的强效抑制剂。
