Stimulation of platelet apoptosis by balhimycin

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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+. Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1 μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca2+, but not on activation of caspases.

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► The antibiotic balhimycin triggers platelet shrinkage and cell membrane scrambling.
► Balhimycin increases cytosolic Ca2+ activity and ceramide formation.
► Balhimycin leads to mitochondrial depolarization and caspase-3 activation.
► Balhimycin induced cell membrane scrambling and caspase activation were Ca2+ dependent.
► Thus, balhimycin triggers platelet apoptosis by increasing Ca2+entry.