Stimulation of Platelet Death by Vancomycin

Abstract

Background/Aims: Side effects of vancomycin, a widely used antibiotic, include thrombocytopenia. The vancomycin-induced thrombocytopenia has been attributed to immune reactions. At least in theory, thrombocytopenia could result in part from the triggering of apoptosis, which results in cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. The cell membrane scrambling could be initiated by a signaling involving increase of cytosolic Ca2+ activity, ceramide formation, mitochondrial depolarization and/or caspase activation. Vancomycin has indeed been shown to trigger neutrophil apoptosis. An effect of vancomycin on platelet apoptosis has, however, never been tested. The present study thus explored the effect of vancomycin on platelet activation and apoptosis. Methods: Human blood platelets were exposed to vancomycin and forward scatter was utilized to estimate cell volume, annexin V-binding to quantify phosphatidylserine (PS) exposure, Fluo-3 AM fluorescence to estimate cytosolic Ca2+ activity ([Ca2+]i), antibodies to quantify ceramide formation and immunofluorescence to quantify protein abundance of active caspase-3. Results: A 30 minutes exposure to vancomycin (≥1 µg/ ml) decreased cell volume, triggered annexin V-binding, increased [Ca2+]i, activated caspase 3, stimulated ceramide formation, triggered release of thromboxane B2, and upregulated surface expression of CD62P (P-selectin) as well as activated integrin αllbβ3. Annexin V-binding and upregulation of CD62P (P-selectin) and integrin αllbβ3 was significantly blunted by removal of extracellular Ca2+. Annexin V-binding was not significantly blunted by pan-caspase inhibitor zVAD-FMK (1 µM). In conclusion, vancomycin results in platelet activation and suicidal platelet death with increase of [Ca2+]i, caspase-3 activation, cell membrane scrambling and cell shrinkage. Activation and cell membrane scrambling required the presence of Ca2+, but not activation of caspases. Conclusion: Vancomycin exposure leads to platelet activation and apoptosis.

Keywords

• Platelets
• Phosphatidylserine
• Caspase-3
• Apoptosis
• Cell membrane scrambling
• Mitochondrial potential
• Ca2+