The proteome of erythrocyte-derived microparticles from plasma: new clues for erythrocyte aging and vesiculation.

Vesicle formation is an integral part of the physiological erythrocyte aging process. Recent biophysical and immunochemical data have suggested that vesicles originate by the extrusion of membrane patches that, during aging, have become damaged and simultaneously enriched in removal signals. Thereby, vesiculation may serve to postpone the untimely removal of functional cells. As a first step toward the identification of the underlying mechanisms, we isolated erythrocyte-derived vesicles from plasma by fluorescence-activated cell sorting, analyzed their proteome by mass spectrometry, and compared this with the membrane proteomes of erythrocytes that were separated according to cell age. The presence of band 3 and actin in the vesicles together with the absence of almost all other integral membrane and cytoskeletal proteins, and the specific, aging-associated alterations in band 3 aggregation and degradation shown by proteomics as well as immunochemistry, all suggest that the erythrocyte aging process harbors a specific, band 3-centered mechanism for vesicle generation. The age-related recruitment of plasma proteins, proteins of the ubiquitin-proteasome system, and small G proteins to the erythrocyte membrane supports the hypothesis that modification of band 3 and/or degradation initiate vesiculation, and the subsequent recognition and fast removal of vesicles by the immune system. This article is part of a Special Issue entitled: Integrated omics.

Susceptibility to hyperosmotic stress-induced phosphatidylserine exposure increases during red blood cell storage.

BACKGROUND: During storage of red blood cell (RBCs) before transfusion, RBCs undergo a series of structural and functional changes that include the exposure of phosphatidylserine (PS), a potent removal signal. It was postulated that, during blood bank storage, the susceptibility to stress-induced PS exposure increases, thereby rendering a considerable fraction of the RBCs susceptible to rapid removal after transfusion. STUDY DESIGN AND METHODS: RBCs were processed and stored following standard Dutch blood bank procedures. Samples were taken every week for up to 6 weeks and exposed to various stress conditions, such as hyperosmotic shock and energy depletion. The effect of these treatments on PS exposure was measured by flow cytometric analysis of annexin V binding. The same analyses were performed on RBCs that had been separated according to density using discontinuous Percoll gradients. RESULTS: During storage under blood bank conditions, RBCs become increasingly susceptible to loss of phospholipid asymmetry induced by hyperosmotic shock and energy depletion. Especially the RBCs of higher densities, that have a smaller volume and an increased HbA1c content as is typical of aged RBCs, become increasingly susceptible with storage time. CONCLUSIONS: During storage, RBCs develop an increased susceptibility to stress-induced loss of phospholipid asymmetry that is especially associated with an aging phenotype. This increased susceptibility may be responsible for the rapid disappearance of a considerable fraction of the RBCs during the first 24 hours after transfusion.

Erythrocyte vesiculation: a self-protective mechanism?

Previous studies demonstrated that 20% of haemoglobin is lost from circulating erythrocytes during their total lifespan by vesiculation. To study whether removal molecules other than membrane-bound haemoglobin were present in erythrocyte-derived vesicles, flow cytometry and immunoblot analysis were employed to examine the presence of phosphatidylserine (PS) and IgG, and senescent cell antigens respectively. It was demonstrated that 67% of glycophorin A-positive vesicles exposed PS, and that half of these vesicles also contained IgG. Immunoblot analysis revealed the presence of a breakdown product of band 3 that reacted with antibodies directed against senescent erythrocyte antigen-associated band 3 sequences. In contrast, only the oldest erythrocytes contained senescent cell antigens and IgG, and only 0.1% of erythrocytes, of all ages, exposed PS. It was concluded that vesiculation constitutes a mechanism for the removal of erythrocyte membrane patches containing removal molecules, thereby postponing the untimely elimination of otherwise healthy erythrocytes. Consequently, these same removal molecules mediate the rapid removal of erythrocyte-derived vesicles from the circulation.

Erythrocyte aging: a more than superficial resemblance to apoptosis?

In physiological circumstances, erythrocyte aging leads to binding of autologous IgG followed by recognition and removal through phagocytosis, mainly by Kupffer cells in the liver. This process is triggered by the appearance of a senescent erythrocyte-specific antigen. The functional and structural characteristics of senescent erythrocytes strongly suggest that this antigen originates on band 3, probably by calcium-induced proteolysis. Generation of vesicles enriched in denatured hemoglobin is an integral part of the erythrocyte aging process. These vesicles are also removed by Kupffer cells, with a major role for exposure of phosphatidylserine. Moreover, senescent erythrocyte-specific antigens are present on vesicles. Thus, vesicles and senescent erythrocytes may be recognized and removed through the same signals. These and other, recent data support the theory that erythrocyte aging is a form of apoptosis that is concentrated in the cell membrane, and provide the context for future studies on initiation and regulation of the erythrocyte aging process. Insight into the normal aging mechanism is essential for understanding the fate of erythrocytes in pathological circumstances and the survival of donor erythrocytes after transfusion.

Liver Kupffer cells rapidly remove red blood cell-derived vesicles from the circulation by scavenger receptors.

Previous studies have shown that during the lifespan of red blood cells (RBCs) 20% of hemoglobin is lost by shedding of hemoglobin-containing vesicles. However, the fate of these vesicles is unknown. To study this fate we used a rat model, after having established that rat RBCs lose hemoglobin in the same way as human RBCs, and that RBC-derived vesicles are preferentially labeled by Na2(51) CrO4. Such labeled vesicles were injected into recipient rats. Within 5 minutes, 80% of the radioactivity was cleared from the circulation with a concomitant uptake by the liver of 55% of the injected dose. After 30 minutes, Kupffer cells contained considerable amounts of hemoglobin and were shown to be responsible for 92% of the liver uptake. Vesicle clearance from the blood as well as liver uptake were significantly inhibited by preinjection of the scavenger-receptor ligands polyinosinic acid and phosphatidylserine. We conclude that in rats Kupffer cells rapidly remove RBC-derived vesicles from the circulation, mainly by scavenger receptors. The same mechanism is likely to be responsible for the elimination of human RBC vesicles, thereby constituting an important pathway for the breakdown of RBCs in humans.

Hemoglobin loss from erythrocytes in vivo results from spleen-facilitated vesiculation.

Previous studies have shown that approximately 20% of hemoglobin is lost from circulating red blood cells (RBCs), mainly during the second half of the cells' life span. Because hemoglobin-containing vesicles are known to circulate in plasma, these vesicles were isolated. Flow cytometry studies showed that most RBC-derived vesicles contain hemoglobin with all hemoglobin components present. The hemoglobin composition of the vesicles resembled that of old RBCs. RBC cohort studies using isotope-labeled glycine have been described, which showed a continuous presence of this label in hemoglobin degradation products. The label concentration of these products increased during the second half of the RBC life span, accompanied by a decrease within the RBC. It is concluded that the hemoglobin loss from circulating RBCs of all ages can be explained by shedding hemoglobin-containing vesicles. This loss occurs predominantly in older RBCs. Apparently the spleen facilitates this process since asplenia vesicle retention within RBCs of all ages has been described, accompanied by an increase in the percentage of total HbA(1). The present study shows that in old RBCs of asplenic individuals, the decrease of hemoglobin content per cell such as seen in old RBCs of control individuals is absent due to an increase in the absolute amount of HbA(1c) and HbA(1e2). It is concluded that hemoglobin-containing vesicles within old RBCs are "pitted" by the spleen.