Cao Hang, Bissinger Rosi, Umbach Anja T, Al Mamun Bhuyan A, Lang Florian, Gawaz Meinrad
Cell Physiol Biochem. 2017;41(1):369-380. doi: 10.1159/000456319. Epub 2017 Jan 26.
BACKGROUND/AIMS: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets.
Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αβ integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE.
Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+], P-selectin abundance, active αβ integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+] both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+], P-selectin abundance, and active αβ integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation.
Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation.
背景/目的：8-氨基喹啉他非诺喹已被证明对疟原虫、利什曼原虫和锥虫有效。该物质至少部分通过触发寄生虫凋亡发挥作用。此外，他非诺喹已被证明可触发红细胞凋亡，即自杀性红细胞死亡，其特征为细胞收缩和细胞膜磷脂酰丝氨酸易位导致的细胞膜紊乱。他非诺喹对红细胞凋亡的影响部分归因于对钙离子内流和氧化应激的刺激。钙离子内流是凝血酶和胶原相关肽（CRP）激活血小板的关键事件。本研究探讨了他非诺喹是否会影响血小板的钙离子内流、激活和凋亡。
将从野生型小鼠分离的血小板暴露于他非诺喹（2.5μg/ml）30分钟，有无凝血酶（0.01U/ml）或CRP（2μg/ml或5μg/ml）的额外处理。采用流式细胞术从Fluo-3荧光估计胞质钙离子活性（[Ca2+]），从P-选择素丰度估计血小板脱颗粒，从αβ整合素丰度估计整合素激活，从膜联蛋白-V结合估计磷脂酰丝氨酸丰度，从前向散射估计相对血小板体积，从DCF荧光估计活性氧（ROS），用活性半胱天冬酶-3染色试剂盒检测半胱天冬酶3活性，并用CD9-APC和CD9-PE染色检测聚集情况。
凝血酶（0.01U/ml）和CRP（2μg/ml或5μg/ml）均显著增加[Ca2+]、P-选择素丰度、活性αβ整合素和膜联蛋白-V结合，且均显著降低血小板体积、激活半胱天冬酶3并刺激聚集。在不存在和存在凝血酶及CRP的情况下，给予他非诺喹（2.5μg/ml，30分钟）均显著降低[Ca2+]。他非诺喹显著减弱凝血酶和CRP对[Ca2+]、P-选择素丰度和活性αβ整合素的影响，但显著增加ROS和膜联蛋白-V结合，显著增强凝血酶对半胱天冬酶3活性和血小板体积的影响，并显著增强血小板聚集。
他非诺喹可抵消凝血酶和CRP诱导的胞质钙离子活性增加和血小板激活，但增强血小板凋亡和血小板聚集。
