Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction

Abstract

Background Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients. Study Design Five RBC units were drawn and divided (half pre-storage leukoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry. Results In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leukoreduction removed many platelet- and leukocyte-derived structural proteins; however, a number of intracellular proteins accumulated including: peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C. We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leukoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2

“…Proteomics on the field blood (plasma) from 2 patients with blunt trauma who later developed ALI/MOF and the plasma from 4 healthy control donors were completed using 2-dimensional gel electrophoresis and mass spectroscopy with computer analyses of the resultant peptides specific for α-enolase (7). …”

“…Repeat ANOVA was performed followed by Neuman-Keuls post-hoc analysis, which was dependent upon the equality of variance. Statistical significance was determined at the p<0.05 level (7). …”

“…In a prospective clinical study, controlled for the amount transfused, transfusion of stored RBCs, versus fresh, was an independent predictor for the development of ALI/MOF in injured patients (5;6). Although some of the pro-inflammatory mediators responsible for ALI/MOF have been identified, others have remained elusive and potential candidates include intracellular proteins that have protease activity similar to thrombin and accumulate during routine storage (7). Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of proteinase-activated receptors (PARs), specifically PAR-1, PAR-3, and PAR-4, through cleavage of the N-terminal exodomain of the receptor, releasing the tethered ligand (8).…”

Experimental prestorage filtration removes antibodies and decreases lipids in RBC supernatants mitigating TRALI in vivo

“…35 We have previously reported that both 5-lipoxygenase and 5-lipoxygenase activating protein are present in RBC units, and their activity likely results in the accumulation of 5-HETE. 36 In addition, there is 12% to 17% sequence homology between human IgG and 5-lipoxygenase activating protein or human IgG and 5-lipoxygenase, which may account for clearance of one of these enzymes by experimental filtration. 37 The PMN priming activity that accumulates in the supernatant during LR-RBC storage is almost totally composed of lipophilic compounds, which were significantly decreased by experimental filtration.…”

Age of blood

“…Supernatant of these RBCs also led to significantly higher increase in CD11b expression on freshly isolated neutrophils. Dzieciatkowska et al [6] analysed supernatant of five RBCs in AS-5 that were split with one part pre-storage leucocyte reduced (or leucoreduced). Proteomic analyses of the supernatant were performed on days 1 and 42.…”