Background:

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 thrombocytopenia, which is at least in part due to triggering of platelet apoptosis, 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.

Methods:

Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phoshpatidylserine 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.

Results:

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).

Conclusions:

Balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca²⁺ , but not on activation of caspases.