Human Platelets Stored at Room Temperature Demonstrate Diminished Resistance to Oxidative Stress Compared to Those Stored at 4°C
Authors: Fu Xiaoyun , Osborn Warren , Shen Yu , Wang Yi , Miles Jeffrey , Fang Lydia , Osborne Barbara , Chen Junmei , López José A. , Stolla Moritz 
Published: February 2021

Background: Current blood banking practices dictate that platelets can only be stored at room temperature (RT) for up to seven days because of functional decline and risk of bacterial overgrowth. A lower storage temperature has the potential to slow platelet metabolism and reduce oxidative damage during storage. In the current study, we quantified by mass spectrometry small molecule thiols and disulfides as markers of oxidative stress in platelets stored in plasma at RT or 4°C. Because glutathione (GSH), γGlu-Cys-Gly, is the major thiol in platelets, we also analyzed metabolites (amino acids and dipeptides) involved in GSH synthesis and degradation cycles.
Method: Platelets were acquired from five healthy donors and adjusted to 3 × 1011/L in plasma. The platelets from each donor were split into two bags and stored in plasma at RT or 4°C (with or without agitation, respectively) for up to 14 days. Aliquots from each group were taken at days 0, 3, 7, and 14, and N-ethylamaleimide (NEM) was added to block free thiols before storing the samples at -80°C until analysis. Total and reduced forms of GSH and cysteine including protein-bound disulfides were quantified by mass spectrometry as described previously [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]. Paired t-tests were used to evaluate the differences between the groups.
Results: GSH is the major intracellular antioxidant that maintains a reducing environment within cells. After 7 days of storage at RT, reduced GSH decreased drastically from 13.0±1.2 pre-storage to 1.7±0.6 µM (p &lt;0.005), an 85% decrease. In contrast, GSH in platelets stored at 4°C decreased at a much slower rate, with over 60% and 50% GSH remaining after 7 and 14 days of storage, respectively, indicating that platelets stored at 4°C maintain higher reducing capacities than those stored at RT. The decrease in reduced GSH can be caused by oxidation and/or degradation. If it is primarily due to oxidation, total GSH ― the sum of reduced and oxidized (disulfides) forms ― should remain relatively constant. The total GSH in the stored platelets decreased in a manner similar to the reduced GSH at both RT and 4°C, suggesting that degradation, not oxidation, was responsible for the decrease in reduced GSH. We then determined the quantities of metabolites related to GSH synthesis and degradation and found significant increases in the total amounts of the amino acids Glu, Cys, and Gly, suggesting that more GSH was degraded than synthesized in platelets during storage. We also examined the changes of Cys during storage at RT and 4°C because Cys is the main thiol in plasma and the platelets were stored in 100% plasma. Protein-bound Cys was significantly elevated in the samples stored at RT compared to those stored at 4°C, the differences being observed as early as day 3 and continuing to day 14 day (p&lt;0.01). We previously identified protein-bound Cys as a marker for oxidative stress in plasma [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]; its elevation is therefore indicative of increased oxidation of the platelet sample during RT storage compared to 4°C storage.
Summary: In this study, we found that the ability of platelets to withstand oxidative stress during storage is severely compromised in platelets stored at RT as opposed to 4°C. This storage lesion affected both intracellular (GSH) and extracellular (Cys) antioxidant molecules and involved 2 mechanisms: GSH degradation and Cys oxidation. We are currently attempting to determine whether the differences observed result from increased metabolic rate in the RT-stored platelets or from greater exposure to ambient air caused by the need for agitation. These studies will identify targets for improving the quality and shelf-life of platelets stored for transfusion.

Disclosures
No relevant conflicts of interest to declare.