Phosphatidylethanolamine is progressively exposed in RBCs during storage

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Red blood cell (RBC) transfusions are a mainstay of modern medicine. However, differences in unit efficacy and safety during storage exist (Rohde et al., 2014; Glynn et al., 2016). Although prospective randomised trials have been performed, there remains some debate on the specific clinical contexts where red cell storage age influences patient outcomes (Glynn et al., 2016).
Currently, additive solutions (AS) preserve RBCs such that if transfused just prior to the expiration date, about 75% of RBCs are recovered 24 h post‐transfusion (Glynn et al., 2016). The mechanism mediating the rapid removal of roughly 25% of stored RBCs is not entirely understood. Well‐documented intracellular changes with stored RBCs include depletion of glucose, ATP and 2,3‐diphosphoglycerate (DPG) (Hess, 2014). Changes to the overall make‐up of the unit also occur, including increases in cell‐free haemoglobin (both soluble and microparticle‐encapsulated), potassium and acids (Donadee et al., 2011). More recent studies have highlighted a critical role for nitric oxide (NO) in the changes that occur during RBC storage, including depletion of S‐nitrosohaemoglobin (Bennett‐Guerrero et al., 2007) and enhanced scavenging of NO (Donadee et al., 2011). These aberrations at the molecular level are further associated with cellular‐level defects including red cell morphology changes, decreased red cell deformability and membrane bilayer disruption including the formation of microparticles (MP) (Berezina et al., 2002).
The red cell lipid bilayer is composed of phosphatidylcholine, sphingomyelin, phosphatidylserine (PS), phosphatidylinositol, phosphatidic acid and phosphatidylethanolamine (PE) (Virtanen et al., 1998). Under normal conditions, enzymes known as flippases and floppases maintain a controlled lipid asymmetry, with phosphatidylcholine and sphingomyelin predominantly on the outer leaflet, and PS, phosphatidylinositol, phosphatidic acid and PE predominantly on the inner leaflet (Virtanen et al., 1998). Multiple studies have established that the normal red cell membrane asymmetry decreases with storage, with reports of significant increases in PS exposure on the outer leaflet of the cell membrane (Berezina et al., 2002). In quantifying membrane disturbance over the storage duration, studies variably report up to 6% of RBCs exposed PS on the outer leaflets of the lipid bilayer at 42 days (Koshkaryev et al., 2009). This association is of interest, as both human and murine macrophages can bind to and engulf symmetric red cell ghosts, red cells with PS inserted externally, oxidised red cells and sickled red cells, all of which expose PS on the outer leaflet (Styles et al., 1997; Fadok et al., 2001; Zenarruzabeitia et al., 2015). While promising, it remains unclear whether PS exposure on the outer leaflet can entirely explain the rapid phagocytosis of nearly one‐quarter of the red cells in an aged stored unit post‐transfusion.
We recently found that PE is also exposed on the outer leaflet of crenated/damaged erythrocytes in aged RBCs (Larson et al., 2012). Unlike PS, which makes up only 3–15% of total lipids, PE is the second‐most abundant phospholipid in mammalian cells, making up 20–45% of total lipids (Zhao, 2011). Thus, significantly increased PE exposure on outer leaflet may also contribute to the extensive removal of older stored red cells.
To determine whether storage age is associated with increased PE exposure on RBC surface, we performed a longitudinal evaluation of PE using the novel PE‐probe, duramycin. Duramycin is a well‐described lantibiotic that binds to PE with high affinity (Zhao, 2011), and, as reported previously (Larson et al., 2012), can be used as a sensitive and specific marker for PE content on the outer leaflet of the red cells.
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