Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

Abstract

© F e r r a t a S t o r t i F o u n d a t i o ndifferent genetic and clinical status provided new insights into the pathogenic role of circulating MP and possible interventions to control their amount in thalassemia. MethodsUnless otherwise stated, all materials were obtained from Sigma-Aldrich, St. Louis, MO, USA. Additional information about the methods are provided in the Online Supplementary Data. Treatment of red blood cellsVenous blood was drawn from 12 healthy individuals, eight non-splenectomized TI patients and six splenectomized TI patients. All subjects provided written, informed consent before entering the study. The study was approved by the local Ethics Committee and conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.Clinical analyses were performed by routine laboratory tests at the Policlinico Hospital (Milan, Italy). Blood anti-coagulated with heparin was stored in citrate-phosphate-dextrose with adenine (CPDA-1) prior to its use. RBC were washed three times with phosphate-buffered saline (PBS: 137mM NaCl, 2.7mM KCl, 8.1mM K 2 HPO 4 , 1.5mM KH 2 PO 4 , pH 7.4) containing glucose 5 mM to obtain packed RBC.To stimulate HMC formation, RBC from healthy donors were suspended at a hematocrit of 30% and incubated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 mM) of phenylhydrazine at 37 °C for 4 h. To test the antioxidant effect in MP release we incubated 200 μM of 2-mercaptoethanol in the presence of 1 mM phenylhydrazine. When necessary, RBC were pretreated with Syk kinase inhibitors (10 μM Syk inhibitors II and 10 μM Syk inhibitors IV, Calbiochem, Darmstadt, Germany), for 1 h at 37 °C in the dark. Each reaction was terminated by three washes with PBS-glucose. For all protocols described, untreated controls were processed identically except that the stimulant/inducer was omitted from the incubation. Red blood cell membrane preparationStandard hypotonic membranes were prepared, as previously described, 20 and stored frozen at -80°C until use. Membrane protein content was quantified using the DC Protein assay (Biorad, Hercules, CA, USA). Analysis of microparticlesThe MP in plasma were analyzed by flow cytometry using a modification of a previously described method:22 25 μL of plasma diluted 1:1 with PBS-glucose 5mM were analyzed using anti-CD41 (BD, Franklin Lakes, NJ, USA) and anti-glycophorin-A (Dako, Denmark), both diluted 1:10. Assay of hemichromesHMC were quantified by measuring heme absorbance (Abs) using the following equation:HMC= -133xAbs577 -114xAbs630 + 233xAbs560, and expressed as nMoles/mL of solubilized membranes. 23 Microparticle isolationTo induce MP release in vitro, RBC from each volunteer and phenylhydrazine-treated RBC in PBS (30% hematocrit) were incubated as previously described. 24 Electrophoresis and immunoblottingMembrane and MP proteins were solubilized in Laemmli buffer 25 under reducing [2% (w/v) dithiothreitol] or non-reducing conditions at a volume ratio of 1:1. Sodium dodecylsulfate polyacrylamide gel electropho...

Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/β-thalassemic patients

“…We observed that antioxidant proteins, chaperone proteins, proteins involving in iron metabolism, hemoglobin subunit d, cathepsin S (an inflammatory marker), and erythroid proteins were consistently increased in quantity in HbE/ b-thalassemic patients across all 3 pooled samples (Table 2), and this was also observed in 6 individual samples (Table 4). Taken together, the observed alterations in protein content in the thalassemic EVs are consistent with the known increase in oxidative burden due to peripheral hemolysis reported in previous studies, 13,22,24 and this study has substantially extended the number of known proteins with an altered concentration in HbE/ b-thalassemic patients' EVs.…”

“…13 In our study, we detected these proteins in at least twofold greater abundance in thalassemic EVs when compared with EVs from control individuals. The presence of these antioxidant proteins likely reflects the stressresponse mechanism in thalassemic erythrocytes, and we propose this could be a result of either EVs shedding from the viable erythrocytes or being generated from red cell lysis.…”

“…12 Previous studies of the proteomics profile in EVs in the plasma released from the platelets and RBCs of b-thalassemic patients reported a higher level of proteins involved in the oxidative stress response, for example, catalase, peroxiredoxin 2 (PRDX2), and heat shock proteins 70 (Hsp70) when compared with healthy individuals. 13,14 In addition, evidence of increased RBC generation in b-thalassemia was identified, such as detection of m hemoglobin, which is transcribed from the a gene (HBM) and usually expressed in cord blood reticulocytes. 15 The close association between EV generation and pathophysiology of HbE/b-thalassemia suggests that the severity spectrum of clinical manifestations of the patients might be shown in the composition of EVs.…”

Physiology and pathology of extracellular vesicules

“…1. Вид эритроцитарных микровезикул из плазмы крови пациента с бета-талассемией при наблюдении в световой микроскоп (воспроизведе-но из [8]): а -флуоресцентная конфокальная микроскопия, б -классическая световая микроскопия. Шкала масштаба -1 µм Fig.…”

“…1. Erythrocyte microvesicles from plasma of a patient with beta-talassemia as seen in an optical microscope (reproduced from [8]): a -fluorescence confocal microscopy, б -traditional light microscopy. Scale -µm…”