Department of Physiology, Eberhard-Karls-University Tuebingen, Tuebingen, Germany.
Biochem Biophys Res Commun. 2013 May 31;435(2):323-6. doi: 10.1016/j.bbrc.2013.01.120. Epub 2013 Feb 9.
Glycopeptides, such as vancomycin, are powerful antibiotics against methicillin-resistant Staphylococcus aureus. Balhimycin, a glycopeptide antibiotic isolated from Amycolatopsis balhimycina, is similarly effective as vancomycin. Side effects of vancomycin include triggering of platelet apoptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulation of apoptosis may involve increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization and/or caspase activation. An effect of balhimycin on apoptosis has, however, never been reported. The present study thus tested whether balhimycin triggers platelet apoptosis. Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V-binding, cytosolic Ca(2+) activity from fluo-3AM fluorescence, ceramide formation utilizing antibodies, mitochondrial potential from DiOC6 fluorescence, and caspase-3 activity utilizing antibodies. As a result, a 30 min exposure to balhimycin significantly decreased cell volume (≥1 μg/ml), triggered annexin V binding (≥1 μg/ml), increased cytosolic Ca(2+) activity (≥1 μg/ml), stimulated ceramide formation (≥10 μg/ml), depolarized mitochondria (≥1 μg/ml) and activated caspase-3 (≥1 μg/ml). Cell membrane scrambling and caspase-3 activation were virtually abrogated by removal of extracellular Ca(2+). Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca(2+), but not on activation of caspases.
糖肽类抗生素，如万古霉素，是治疗耐甲氧西林金黄色葡萄球菌的有效抗生素。巴尔希霉素是从糖多孢菌中分离得到的糖肽类抗生素，与万古霉素同样有效。万古霉素的副作用包括触发血小板凋亡，其特征是细胞收缩和细胞膜翻转，磷脂酰丝氨酸暴露在细胞表面。凋亡的刺激可能涉及细胞浆钙离子活性增加、神经酰胺形成、线粒体去极化和/或半胱氨酸蛋白酶激活。然而，巴尔希霉素对凋亡的影响从未被报道过。因此，本研究检测了巴尔希霉素是否触发血小板凋亡。用人血血小板处理巴尔希霉素，通过前向散射估计细胞体积，通过 annexin V 结合检测磷脂酰丝氨酸暴露，通过 fluo-3AM 荧光检测细胞浆钙离子活性，通过抗体检测神经酰胺形成，通过 DiOC6 荧光检测线粒体电位，通过抗体检测半胱氨酸蛋白酶-3 活性。结果显示，30 分钟暴露于巴尔希霉素可显著降低细胞体积（≥1μg/ml），触发 annexin V 结合（≥1μg/ml），增加细胞浆钙离子活性（≥1μg/ml），刺激神经酰胺形成（≥10μg/ml），使线粒体去极化（≥1μg/ml）并激活半胱氨酸蛋白酶-3（≥1μg/ml）。去除细胞外钙离子可显著阻断细胞膜翻转和半胱氨酸蛋白酶-3 激活。用 zVAD-FMK（1μM）抑制全半胱氨酸蛋白酶不能显著削弱细胞膜翻转。总之，巴尔希霉素触发血小板细胞膜翻转，这一效应依赖于钙离子，但不依赖于半胱氨酸蛋白酶的激活。
