Characterizations and proteome analysis of platelet-free plasma-derived microparticles in β-thalassemia/hemoglobin E patients

Chaichompoo, Pornthip; Kumya, Panida; Khowawisetsut, Ladawan; Chiangjong, Wararat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta; Sirithanaratanakul, Noppadol; Fucharoen, Suthat; Thongboonkerd, Visith; Pattanapanyasat, Kovit

doi:10.1016/j.jprot.2012.06.004

Cited by 43 publications

(64 citation statements)

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“…The presence of MP generated from many cell types poses further methodological limitations to the analysis of circulating MP. 32 In accordance with a study performed in patients with HbE, 33 plasma proteins were the major proteins identified in MP isolated from patients' plasma and we were able to identify only α−globin chains and band 3 as RBC components.…”

Section: E Ferru Et Alsupporting

confidence: 87%

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

Ferru

Pantaleo

Carta³

et al. 2013

Haematologica

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© 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...

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Section: E Ferru Et Alsupporting

confidence: 87%

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

Ferru

Pantaleo

Carta³

et al. 2013

Haematologica

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“…Inflammatory cytokines appear to play a role in ineffective erythropoiesis (26). Moreover, platelet hyperactivity and platelet-RBC interactions, as discussed above, coupled with increased levels of specific cytokines in HbE/b-thal patients partake in signaling hypererythropoiesis and set the stage for inflammation as an upstream pathophysiological mechanism in this disease (31,90).…”

Section: Fig 2 Hbe Acts As a Bmentioning

confidence: 99%

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

Hirsch

Sibmooh

Fucharoen

et al. 2017

Antioxidants & Redox Signaling

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Significance: Oxidative stress and generation of free radicals are fundamental in initiating pathophysiological mechanisms leading to an inflammatory cascade resulting in high rates of morbidity and death from many inherited point mutation-derived hemoglobinopathies. Hemoglobin (Hb)E is the most common point mutation worldwide. The b E -globin gene is found in greatest frequency in Southeast Asia, including Thailand, Malaysia, Indonesia, Vietnam, Cambodia, and Laos. With the wave of worldwide migration, it is entering the gene pool of diverse populations with greater consequences than expected. Critical Issues: While HbE by itself presents as a mild anemia and a single gene for b-thalassemia is not serious, it remains unexplained why HbE/b-thalassemia (HbE/b-thal) is a grave disease with high morbidity and mortality. Patients often exhibit defective physical development, severe chronic anemia, and often die of cardiovascular disease and severe infections. Recent Advances: This article presents an overview of HbE/b-thal disease with an emphasis on new findings pointing to pathophysiological mechanisms derived from and initiated by the dysfunctional property of HbE as a reduced nitrite reductase concomitant with excess a-chains exacerbating unstable HbE, leading to a combination of nitric oxide imbalance, oxidative stress, and proinflammatory events. Future Directions: Additionally, we present new therapeutic strategies that are based on the emerging molecular-level understanding of the pathophysiology of this and other hemoglobinopathies. These strategies are designed to short-circuit the inflammatory cascade leading to devastating chronic morbidity and fatal consequences. Antioxid. Redox Signal. 26,[794][795][796][797][798][799][800][801][802][803][804][805][806][807][808][809][810][811][812][813]

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“…The GPA/CD45 stained leukocytes and erythrocytes from K 3 EDTA peripheral blood samples were determined for ROS as described in the previous study [17]. Briefly, samples were incubated with 0.4 mM DCFH-DA dye for 15 min at 37°C in a humidified incubator with 5% CO 2 .…”

Section: Oxidative Stress Analysismentioning

confidence: 99%

Accelerated telomere shortening in β-thalassemia/HbE patients

Chaichompoo

Pattanapanyasat

Winichagoon

et al. 2015

Blood Cells, Molecules, and Diseases

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Characterizations and proteome analysis of platelet-free plasma-derived microparticles in β-thalassemia/hemoglobin E patients

Cited by 43 publications

References 32 publications

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

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

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

Accelerated telomere shortening in β-thalassemia/HbE patients

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