Changes in Band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells

Abstract

BACKGROUND Lipid loss in the form of vesicles contributes to the red blood cell (RBC) storage lesion, and this loss of lipid is correlated with changes in membrane protein function. Sensitive spectroscopic techniques were used to measure changes in Band 3 oligomeric state during storage of RBCs, compared to metabolic changes and phospholipid loss. The aim of the study was to determine whether changes in the macromolecular organization of membrane proteins occur before, coincident with, or after lipid loss during RBC storage. STUDY DESIGN AND METHODS Five RBC units were collected from normal volunteers and stored under standard blood bank conditions, and both metabolic changes and lipid loss were measured by multiple assays. Band 3 oligomeric state was assessed by time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer of eosin-5-maleimide–labeled RBC ghosts. RESULTS Extracellular pH decreased and extracellular potassium increased rapidly during cold storage of blood. Band 3 on the RBC membrane exhibited a shift from small to large oligomers early in the storage period and before detectable loss of phospholipid from the RBC membrane. The immobilized fraction of Band 3, that which is tethered to the cytoskeletal network via spectrin and ankyrin, did not change during cold storage. CONCLUSION Our results demonstrate that changes in the macromolecular organization of membrane proteins on the RBC occur early in storage, and these changes may induce phospholipid loss, irreversible morphologic changes, and loss of function during RBC storage.

Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage

“…This latter has been recently demonstrated to play a regulatory role in the interactions between membrane and cytoskeleton by reducing the band 3 affinity for ankyrin, with consequent enhancement of the lateral mobility of band 3 in the bilayer and progressive vesiculation . However, Karon et al demonstrated that the immobile fraction of band 3 tethered to spectrin cytoskeleton via ankyrin does not change during RBC cold storage . This observation seems to suggest the maintenance of an intact band 3‐ankyrin‐spectrin bridge during storage under blood banking conditions and the presence of some other process for altered band 3 organization, not relying on the Tyr phosphorylation status, which may lead to storage‐related vesiculation and loss of phospholipid from the erythrocyte membrane…”

Molecular mechanisms of erythrocyte aging

“…As erythrocytes age in storage, they metabolize glucose and generate lactate and protons as end products. These initial changes are seen rapidly, often in the first 2 weeks of cold storage (Karon et al, 2009). Excessive 2,3-diphosphoglycerate interacts with membrane proteins, causing structural changes, and also impacts the erythrocyte’s ability to deliver oxygen to the tissues (Tsai et al, 2004; Flatt et al, 2014).…”

“…Formation of the spectrin-actin-protein 4.1 complex decreases during storage (Wolfe et al, 1986). Band 3, usually freely mobile, aggregates into large oligomers that may result in phospholipid loss (Kriebardis et al, 2007; Karon et al, 2009). This is the result of both protein loss and oxidative cross-linking.…”

“…The cumulative result of these changes is that aging erythrocytes lose membrane domain as well as their classic biconcave disc shape, subsequently changing to echinocytes and spherocytes with a loss of their normal deformability (Figure 1) (Izzo et al, 1999; Bennett-Guerrero et al, 2007; Relevy et al, 2008; Karon et al, 2009; Hess, 2010b). Phosphatidylserine, normally on the intracellular side of the plasma membrane, becomes externalized (Hess, 2010b).…”

Beneficial Effects of Melatonin on Oxidative Damage Observed During Whole Blood Storage.

“…6,7 The most dramatic changes that occur during storage include acidosis, 8,9 loss of function of cation pumps and consequent loss of intracellular potassium 10,11 and increased oxidative stress. [11][12][13] An increase in oxidative stress during storage results in lipid peroxidation in which reactive oxygen species (ROS) attack the membrane and lead to loss of membrane integrity and cell death. 14 To counter this potential damage, cells have antioxidant defense mechanisms of enzymatic (superoxide dismutase (SOD), catalase) 15 and nonenzymatic (tocopherols, ascorbic acid) types.…”