Deoxygenation of leucofiltered erythrocyte concentrates preserves proteome stability during storage in the blood bank.

Abstract

While prospective clinical trials are currently underway in several countries throughout the world1, the safety and effectiveness of long-stored erythrocyte concentrates have been questioned by controversial retrospective clinical evidence2,3 and accumulating biochemical investigations4–6. Laboratory investigations have recently highlighted that a significant number of biochemical and mechanical lesions accumulate in erythrocyte concentrates during their storage in the blood bank. These alterations, often referred to as “storage lesions”, include: (i) the progressive impairment of cell metabolism, leading to the consumption of high energy phosphate compounds (ATP and 2,3-diphosphoglycerate)7; (ii) the accumulation of oxidative stress, resulting in the oxidation8, non-enzymatic glycosylation9, fragmentation or aggregation of the red blood cell (RBC) membrane10–13 and cytosolic proteins14; (iii) the progressive loss of metabolic modulation15, as a result of oxidative stress (especially in non-leucoreduced units)16 and protease activity-dependent fragmentation of the N-terminal cytosolic domain of band 317, which results in impairment of the regulatory function of the so-called “respiratory metabolon”18,19; (iv) the progressive oxidation of lipids and deregulation of lipid homeostasis20,21; (v) the exacerbation of membrane blebbing and shedding of microvesicles, a self-protective mechanism22, closely mimicking apoptosis23, which allows cells to get rid of no-longer functional or irreversibly altered proteins and lipids24, while retaining rheological properties of the donor25, which might result in pro-immunogenic potential in the recipient, together with newly appearing storage-dependent biomarkers promoting RBC phagocytosis, such as phosphatidylserine exposure; and (vi) microvesiculation events that compromise RBC morphology (changes from a discocytic to a spheroechnicocytic or spherocytic phenotype), which ends up impairing the surface-to-volume ratio, thereby increasing osmotic fragility26,27, and increasing membrane rigidity, as a result of progressive leaching and intercalation of the plasticizers (such as DEHP) into the membrane lipid bilayer28. In the light of such changes, alternative storage strategies have been proposed over the years, such as deoxygenation of packed erythrocyte concentrates through other methods29,30. The rationale underpinning such a strategy is that deoxygenation of erythrocyte concentrates would remove the main substrate for the production of reactive oxygen species, while promoting energy metabolism through the Embden-Meyerhoff pathway in the light of the oxygen-dependent metabolic modulation mediated by the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 318,19. Early studies by independent groups (and through independent processing strategies29,30) found deoxygenation-dependent improvement of hemolysis and morphological parameters, as well as prolonged preservation of high energy phosphate compounds29–31, though at the expenses of the NADPH-generating potential via the pentose phosphate pathway, as gleaned through metabolomics approaches31. In the light of these encouraging results, we decided to apply a proteomics workflow to understand whether the RBC membrane proteome of erythrocytes stored under deoxygenation in leucofiltered-units is better preserved than in untreated controls. In our previous investigation on deoxygenated units of non-leucofiltered erythrocyte concentrates12 we concluded that the overall spot numbers in two-dimensional electrophoresis (2DE) gels could represent a diagnostic marker of proteome homeostasis during storage in the blood bank. Indeed, RBC are devoid of nuclei and organelles and are, therefore, incapable of synthesising proteins de novo. Any alteration to the overall number of spots in 2DE gels can, therefore, be attributed to storage-dependent effects on the membrane proteome, which we previously identified as fragmentation and aggregation phenomena during the first two-three weeks of storage (increased number of detected spots), followed by vesiculation of damaged proteins (decreased number of spots)11,12. Furthermore, in control units, we previously correlated the storage-dependent alteration of the erythrocyte membrane proteome stability to an impairment in osmotic fragility26. Concordantly, in the present study we performed 2DE assays of the membrane proteome of leucofiltered erythrocytes stored under control conditions or in a deoxygenated environment (following oxygen removal, as previously described)30. As a result, we could confirm that deoxygenation had beneficial effects in terms of membrane proteome homeostasis and preserved osmotic resistance.