Calcium-sensitive mitochondrial membrane potential in human platelets and intrinsic signals of cell death

Abstract

The mechanisms involved in storage-induced damage in platelets are not well understood, but membrane signalling via Ca²⁺ ion flux may affect mitochondrial H⁺ gradients and metabolism and the intrinsic pathways of cell death, platelet survival and function. In this study, the effects of blood bank storage conditions, including reduced plasma concentration and interrupted agitation, were evaluated in platelets from 136 healthy donors. Mitochondrial membrane potential (ΔΨ_m), an indicator of intrinsic cell death, and its sensitivity to Ca²⁺ ionophore A23187, were monitored using JC-1 by flow cytometry and fluorescence microscopy. Platelet survival was examined using lactate dehydrogenase release, annexin V binding and caspase-3/7 activity. Decreased plasma concentration and interrupted agitation affected ΔΨ_m and caspase-3/7. Over 7 days in 30% plasma ΔΨ_m showed a significant reduction (86.3 ± 1.1% platelets with polarised mitochondria day 1; 79.9 ± 2.1% day 5; 75.1 ± 3.8% day 7, P = 0.01 day 1 vs. day 7). Whilst ΔΨ_m in agitated platelets in 100% plasma was unchanged up to day 7, interruption of agitation was associated with a 44% reduction in the proportion of platelets with polarised mitochondria after 5 days (56 ± 11%). The Ca²⁺ sensitivity of ΔΨ_m changed earlier: 5 µM A23187 caused a 20–30% change in the fraction of platelets with polarised mitochondria by day 5. Ca²⁺ sensitivity also increased during interrupted agitation and reduced plasma concentration. ΔΨ_m also correlated with indicators of platelet death, caspase-3 activity and annexin V binding (correlation coefficients of 0.8). In conclusion, changes in Ca²⁺-sensitive ΔΨ_m are involved in the initiation of storage-induced cell death signals that influence platelet count and function in vivo.

• Mitochondria
• membrane potential
• human platelet
• calcium
• ageing
• degeneration
• caspase-3/7