Frans L. A. Willekens

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 versicles 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 initation 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.

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.

Comparative proteomics of erythrocyte aging in vivo and in vitro

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.

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.