Marianna H. Antonelou

An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies

Red blood cell (RBC) aging in the blood bank is characterized by the accumulation of a significant number of biochemical and morphologic alterations. Recent mass spectrometry and electron microscopy studies have provided novel insights into the molecular changes underpinning the accumulation of storage lesions to RBCs in the blood bank. Biochemical lesions include altered cation homeostasis, reprogrammed energy, and redox metabolism, which result in the impairment of enzymatic activity and progressive depletion of high‐energy phosphate compounds. These factors contribute to the progressive accumulation of oxidative stress, which in turn promotes oxidative lesions to proteins (carbonylation, fragmentation, hemoglobin glycation) and lipids (peroxidation). Biochemical lesions negatively affect RBC morphology, which is marked by progressive membrane blebbing and vesiculation. These storage lesions contribute to the altered physiology of long‐stored RBCs and promote the rapid clearance of up to one‐fourth of long‐stored RBCs from the recipients bloodstream after 24 hours from administration. While prospective clinical evidence is accumulating, from the present review it emerges that biochemical, morphologic, and omics profiles of stored RBCs have observable changes after approximately 14 days of storage. Future studies will assess whether these in vitro observations might have clinically meaningful effects.

RBC‐derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components

BACKGROUND: Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail.

Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells

Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost‐bound Hb consists of non‐reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb‐membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin‐ and cytoskeleton‐associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

Intracellular Clusterin Inhibits Mitochondrial Apoptosis by Suppressing p53-Activating Stress Signals and Stabilizing the Cytosolic Ku70-Bax Protein Complex

Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown. Experimental Design: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches. Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax. Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation

BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell‐signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period.

Red blood cell aging markers during storage in citrate‐phosphate‐dextrose–saline‐adenine‐glucose‐mannitol

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail.

Aging and death signalling in mature red cells: from basic science to transfusion practice

The red blood cells (RBCs) aging process is considered as an issue of special scientific and clinical interest. It represents a total of unidirectional, time-dependent but not-necessarily linear series of molecular events that finally lead to cell clearance1. Under normal circumstances, all human RBCs live approximately 120±4 days in blood circulation, implying the existence of tightly regulated molecular mechanism(s), responsible for the programming of the lifespan and the nonrandom removal of senescent RBCs2,3. Although the RBCs have already been used as a model for aging study1, the molecular participants, as well as the signalling pathways involved, are not yet completely clarified. RBCs storage under blood bank conditions is far from being considered analogous to the physiologic in vivo aging process. The putative implicated in vivo signalling pathways are expected to be more-or-less preserved under in vitro conditions, nevertheless slightly modulated, in response to a totally different environment. A storage period of up to 35–42 days at 4 °C probably is not a “congenial interlude” of the physiological maturity process and definitely does not represent an ignorable time period, compared to the RBCs lifespan. Stored RBCs age without the normal adjacency of other cells or plasma, which continuously provide them with survival factors and signals and, moreover, they are obligated to share their living space with their own and other cells’ wastes. Since no clearance mechanisms seem to function, senescent RBCs are probably sentenced to “survive” for a longer period than they were probably programmed for. Although there is evidence suggesting that storage disturbs the physiological RBC aging process4–7, the mechanistic basis of the aging progress inside the blood unit and the functional reactivity of the modified RBCs in vivo, remain still elusive. Given the fundamental need for safe and efficient transfusions, the clinical impact of stored blood, as a function of the storage parameters, has attracted considerable attention. Clinical trials that focus on the potential adverse clinical consequences of transfusing older storage-age RBCs units vs. younger ones have already been reported8. Apart from the skepticism around their design and execution9, these studies indicate the necessity of thorough examination of the storage effect on packed RBCs, before analyzing their potential impact on the clinical outcome. This review focuses on the current knowledge on aging and death signalling pathways operating in both in vivo systems and stored RBCs and suggests future directions in the preservation science, helpful for addressing what seem to be the current critical questions in transfusion medicine.

Effects of pre-storage leukoreduction on stored red blood cells signaling: a time-course evaluation from shape to proteome.

The introduction of pre-storage leukoreduction in the preparation of standard RBCs intended for transfusion provided significant improvement in the quality of labile products and their post transfusion viability and effects, although the literature data are controversial. To elucidate the issue of the probable leukoreduction effects on RBCs storage lesion, we evaluated various storage quality measures in RBCs stored in either leukoreduced (L) or non-leukoreduced (N) units, with emphasis to senescence and oxidative stress associated modifications. Our data suggest that the residual leukocytes/platelets of the labile products represent a stressful storage factor, countering the structural and functional integrity of stored RBCs. Hemolysis, irreversible echinocytosis, microvesiculation, removal signaling, ROS/calcium accumulation, band 3-related senescence modifications, membrane proteome stress biomarkers as well as emergence of a senescence phenotype in young RBCs that is disproportionate to their age, are all encountered more or mostly in N-RBCs compared to the L-RBCs, either for a part or for the whole of the storage period. The partial, yet significant, alleviation of so many storage-related manifestations in the L-RBCs compared to the N-RBCs, is presented for the first time and provides a rational mechanistic interpretation of the improved storage quality and transfusions observed by the introduction of pre-storage leukoreduction. This article is part of a Special Issue entitled: Integrated omics.

Cell-derived microparticles in stored blood products: innocent-bystanders or effective mediators of post-transfusion reactions?

Plasma membrane-derived microvesicles or microparticles (MPs) are sub-cellular vesicles released upon shear stress, cell activation, injury or apoptosis. They represent factors of the extracellular vesicular compartment1 that also includes smaller (0.03–0.1 μm) multivascular body-derived exosomes and larger (1–5 μm) apoptotic bodies which may contain fragmented DNA1. Exosomes are small export vesicles initially derived from the plasma membrane by an endocytosis-involving internalization of the later. In contrast, MPs are larger than exosomes and are derived directly from the plasma membrane after local cytoskeleton rearrangements and membrane budding2. Despite different generation mechanisms and effects, they have been considered as universal biomarkers of cell activation, injury or apoptosis, in a broad range of physiological and pathological processes. Typically MPs range in size from 0.1 μm to 1.0 μm. They express surface markers from their parental cells that allow identification of MPs sub-groups according to their origin: from platelets (P-MPs), leukocytes (L-MPs), red blood cells (R-MPs), endothelial cells and other tissue cells. They harbor cell-derived membrane-bound and cytoplasmic proteins (e.g. chaperones) as well as bioactive lipids. However, MPs are not replicas of the maternal cells and plasma membrane, suggesting a level of selectivity in their formation and sorting of cellular proteins to release2,3. In fact, MPs release is a highly controlled process. According to proteomic data, plasma MPs are enriched in Ig μ-chains, J-chains, profilin I and cyclophilin A, suggesting that MPs-bound IgM may provide a mechanism for their clearance4. As mentioned above, MPs can be formed through several induction pathways, which determine their different molecular profiles and biologic activities. A major aspect in cell biology is communication, which occurs through a direct contact between cells or by means of soluble substances, which react with cells. The fact that MPs released by cells influence other cells is a rather new concept but is a basic mechanism to deliver a message in a highly concentrated manner or to add a missing molecule. MPs exert their effects either via stimulation of target cells by receptor interaction or by direct transfer of their contents which can include membrane proteins and lipids, cytoplasmic components of the parent cell or RNA3,5. By this way, MPs may facilitate cell-to-cell interactions and transfer of signals and receptors between different cell types, inducing thus signalling and response in distant cells. Although not characterized in detail, it may be hypothesized that uptake and removal by cells occurs by similar or identical molecular mechanisms1. The importance of cellular communication via MPs is that they contain highly concentrated signalling components and thus the massive hit of every target cells by MPs’ components is likely to be more effective than the activity of individual components in solution2. As a result, MPs represent nowadays a novel mechanism of intercellular communication mediating inflammation, coagulation and immune responses. The rate of steady state release of MPs in the blood of healthy individuals is usually low1, however they are found elevated in a variety of pathologies including many thrombotic and inflammatory conditions. While it is not sure if this increase represents a contributor to or a result of the disease deterioration, it identifies MPs as potential biomarkers6,7. On the other hand, L-MPs participate in angiogenesis and as such, they have been suggested as novel therapeutic tools to reset the angiogenic switch in pathologies with altered angiogenesis6. MPs are inherent part of all blood labile products delivered to transfused patients (Figure 1). They are present and accumulate in cellular (red blood cells RBCs, platelets PLTs) concentrates as well as in fresh frozen plasma (FFP) during storage8. A clinically important issue is that MPs may have pro-coagulant activities. It has been demonstrated that P-MPs have from 50 up to 100-times higher pro-coagulant activity than PLTs9. MPs exhibit tissue factor activity, so they may have a role in initiating blood coagulation4. During MPs release, the normal asymmetric distribution of phospholipids between the two leaflets of the plasma membrane is lost, resulting in the exposure of the anionic phospholipid phosphatidylserine (PS). The later contributes to the pro-coagulant property of MPs since it serves for the assembly of coagulation factors into active complexes for thrombin generation. However, some observations also suggest the existence of MPs without PS externalization, like in the case of P-MPs derived from PLTs-poor plasma10 suggesting that they possess other activities aside from pro-coagulant phospholipid one. In recent years, a growing body of literature has demonstrated an increased incidence of adverse clinical outcomes associated with the transfusion of a large number of units or, potentially, with increased storage time of the units. These events include increased risk of infection, renal failure, respiratory failure, multiple organ failure, and death, particularly in physiologically compromised patient populations11,12. Since MPs accumulate in blood labile products during storage, transfusion of more or “older” units will offer to the recipient higher number of MPs. The transfused MPs could increase the risk of adverse reactions, by inducing a hypercoagulable state leading to thromboembolic complications. Inversely, in other situations requiring blood transfusion, a hypercoagulable state may be useful to diminish or even helping to stop the bleeding. Many clinical studies suggest that transfusions might be immunosuppressive, although these observations are not generally accepted13,14. However, a clinical study indicated that transfusions of RBCs might be responsible for a diminished survival in cancer patients15. Figure 1 Microparticles (MPs) are inherent part of all blood labile products and are concomitantly delivered by transfusion to recipients. A growing body of literature has demonstrated an increased incidence of adverse clinical outcomes associated with the transfusion ... This review outlines the current knowledge on MPs present in blood labile products used for transfusion: their formation, the effect of storage and their probable activities towards both the “storage lesion” progression as well as the post-transfusion effects. Although the current clinical studies offer only indirect manifestations of MPs activity in the recipients, a lot of data suggest that MPs cotransfused with stored cells and plasma may exert various favorable or adverse effects pre- and post-transfusion, but are definitely not bystanders in any step of this clinically important process.

Donor-variation effect on red blood cell storage lesion: A close relationship emerges.

Although the molecular pathways leading to the progressive deterioration of stored red blood cells (RBC storage lesion) and the clinical relevance of storage‐induced changes remain uncertain, substantial donor‐specific variability in RBC performance during storage, and posttransfusion has been established (“donor‐variation effect”). In‐bag hemolysis and numerous properties of the RBC units that may affect transfusion efficacy have proved to be strongly donor‐specific. Donor‐variation effect may lead to the production of highly unequal blood labile products even when similar storage strategy and duration are applied. Genetic, undiagnosed/subclinical medical conditions and lifestyle factors that affect RBC characteristics at baseline, including RBC lifespan, energy metabolism, and sensitivity to oxidative stress, are all likely to influence the storage capacity of individual donors’ cells, although not evident by the donors health or hematological status at blood donation. Consequently, baseline characteristics of the donors, such as membrane peroxiredoxin‐2 and serum uric acid concentration, have been proposed as candidate biomarkers of storage quality. This review article focuses on specific factors that might contribute to the donor‐variation effect and emphasizes the emerging need for using omics‐based technologies in association with in vitro and in vivo transfusion models and clinical trials to discover biomarkers of storage quality and posttransfusion recovery in donor blood.