Development of Impaired Cytochrome C Oxidase Activity In Apheresis Platelets During Storage

With current storage requirements, the shelf life of platelet (PLT) products is largely limited by the development of deleterious in vitro changes associated with overall reduction in therapeutic efficacy collectively known as the “Platelet Storage Lesion” (PSL). PSL is characterized by a number of biochemical changes including lactate accumulation, bicarbonate depletion and a fall in pH. We hypothesize that these changes reflect a state of impaired oxidative phosphorylation associated with increased reliance on anaerobic glycolysis that evolves during PLT storage. In this study we evaluated the function and expression of “Cytochrome C Oxidase” (COX), a key mitochondrial enzyme in oxidative phosphorylation, with relation to several in vitro markers of PSL. The studies were performed in apheresis PLT stored for up to 7 days under standard blood bank conditions.

Apheresis PLT concentrates were collected in 100% plasma using a Trima Cell Separator (CaridianBCT, Lakewood CO) to provide products with at least 3 × 10¹¹ PLT. All products were collected in Trima® storage bags and were stored in a flatbed reciprocal agitator at 22 ± 2°C for 7 days. Multiple standard in vitro assays were performed on Days 0 (baseline measurement), 2, 4 and 7 of storage including PLT count, mean PLT volume, pH, pO₂, pCO₂, bicarbonate, lactate & glucose levels, aggregation and ATP release studies (ADP/Collagen), soluble CD40 ligand levels (supernatant) and total intracellular PLT ATP content. In addition, steady state COX kinetics and protein immunoblotting for COX subunits I and IV, were performed using isolated PLT mitochondria from simultaneously collected samples. Data are reported as mean ± 2 SD (n =10 experiments). One-way Analysis of Variance (ANOVA) and post-hoc Tukey's range test were used to compare data obtained at different time points. Differences were considered statistically significant only if the p value was <0.001 taking into the account that performing such large number of tests in the study increased the probability that a significant p value was incorrectly obtained by random chance.

PLT COX function declined significantly throughout storage (Table). Steady-state levels of COX I and IV remained essentially unchanged. This decrease in COX function paralleled progressive ATP depletion and time-dependent changes that were consistent with the development of the PSL.

During storage of apheresis PLT for 7 days, COX function decreased progressively in association with ATP depletion indicating acquired impairment in oxidative phosphorylation. These findings suggest that bioenergetic failure is associated with PSL. Further studies are required to determine if mitochondrial dysfunction is a cause of PSL, and if it can be prevented or is amenable to therapeutic intervention.

No relevant conflicts of interest to declare.