Storage-induced changes in erythrocyte membrane proteins promote recognition by autoantibodies

Contains fulltext : 109602.pdf (publisher's version ) (Open Access)Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation

Gateway to understanding microparticles: standardized isolation and identification of plasma membrane-derived vesicles

Item does not contain fulltextMicroparticles (MPs) are small plasma membrane-derived vesicles that can expose molecules originating from their parental cells. As vectors of biological information they are likely to play an active role in both homeostasis and pathogenesis, making them promising biomarkers and nanomedicine tools. Therefore, there is an urgent need for standardization of MP isolation and analysis protocols to propel our understanding of MP biology to the next level. Based on current methodology and recent insights, this review proposes an optimized protocol for the isolation and biochemical characterization of MPs

Erythrocyte autoantibody immunoprecipitation of biotinylated erythrocyte vesicles from a 35 day old transfusion unit.

<p>(A) Immunoprecipitation with either plasma from patient No. 2, or a monoclonal antibody against band 3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042250#s2" target="_blank">Materials and Methods</a>). Analysis was performed by SDS-PAGE, followed by detection of biotinylated membrane proteins (red, streptavidin) and band 3 (green, polyclonal rabbit antibody). A protein G bead control was included. (B) Example immunoprecipitation of biotinylated erythrocyte vesicles from a 35 day old transfusion unit using plasma from patient No. 1. Analysis was performed by SDS-PAGE, followed by detection of biotinylated membrane proteins using fluorochrome conjugated streptavidin. The same sample was used for Coomassie blue gel staining and subsequent proteomics analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042250#pone-0042250-t002" target="_blank">Table 2</a>). The gel slice which was excised for proteomic analysis is indicated as slice III (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042250#pone.0042250.s001" target="_blank">Table S1</a>). Numbers indicate approximate molecular weight (kDa). Blots were analyzed using the Odyssey Infrared Imaging System.</p

Summary of proteins identified by proteomics analyses of erythrocyte/vesicle immunoprecipitations using erythrocyte autoantibody-containing plasma of patients 1, 8 and 9, and allogeneic plasma (control).

<p>Summary of proteins identified by proteomics analyses of erythrocyte/vesicle immunoprecipitations using erythrocyte autoantibody-containing plasma of patients 1, 8 and 9, and allogeneic plasma (control).</p

Erythrocyte autoantibody immunoprecipitation of stored erythrocytes.

<p>(A) Immunoprecipitation of 35 day old erythrocytes with erythrocyte autoantibody-containing patient plasma and allogeneic control plasma, using TX100 or RIPA extraction buffer and analyzed by SDS-PAGE under reducing or non-reducing conditions, followed by silver staining. A representative result (patient No. 2) from one out of three patient plasmas is shown. (B) Example of a silver stained gel of an immunoprecipitation of 35 day stored erythrocytes with plasma of patient No. 1. The same sample was used for Coomassie blue gel staining and subsequent proteomics analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042250#pone-0042250-t002" target="_blank">Table 2</a>). Gel slices which were excised for proteomic analyses are indicated as slices I and II (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042250#pone.0042250.s001" target="_blank">Table S1</a>). Numbers indicate molecular weight (kDa). Heavy [H] and light [L] antibody chains are indicated by arrows.</p

Summary of patient information.

<p>DAT  =  direct antiglobulin test, IAT  =  indirect antiglobulin test (bovine) titer, AIHA  =  autoimmune hemolytic anemia, AML  =  acute myeloid leukemia, NS =  non-specific, APLS  =  antiphospholipid syndrome. All patients had a positive DAT and IAT.</p

Erythrocyte autoantibody immunoprecipitation of erythrocytes sampled at regular time intervals during storage.

<p>Analysis was performed by SDS-PAGE, followed by silver staining. (A) Protein patterns of precipitates obtained using Ringer, autologous plasma, and a representative example from one out of three allogeneic plasmas, and one out of six autoantibody-containing plasmas (patient No. 2). For the allogeneic controls, day 14 is missing. (B) Mean optical density (OD) of patient (•, solid line, N = 6 patients) and allogeneic control plasma (▴, dotted line, N = 3 volunteers) precipitations. Numbers indicate approximate molecular weight (kDa). Error bars represent standard error, *<i>p</i><0.05.</p

Pedigree and RBC morphology of the subjects in this study.

<p>(A) Patients who are clinically diagnosed with PKAN are indicated in black, the healthy relatives are indicated in white. (B) Representative pictures of blood films used for classification of cell shape. (C) Cells are classified in discocyte, echinocyte, acanthocyte or otherwise misshapen. The graph depicts the percentages of different cell morphologies in PKAN patients (marked with an asterisk) and their unaffected family members, compared to healthy donors (control 1+2) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.s001" target="_blank">S1 Fig</a> for other PKAN families).</p

Measurements of fetal haemoglobin (HbF) in red cells of PKAN patients and their relatives.

<p>Black, patients; grey, controls (Ctrls) and family members. The control value is the mean of 6 control samples. The error bar depicts the standard deviation.</p

Microparticle formation by PKAN RBCs as characterized by flow cytometry.

<p>(A) The amount of RBC MPs in plasma. (B) Membrane composition of in vitro generated MPs. Band 3 and glycophorin A (GpA) was measured on MPs collected after 4 and 24h incubation of the RBCs in Ringer solution at 37 °C.</p