The Oxidative Status of Valinomycin-Resistant Normal and Thalassemic Red Cells (RBCs).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3191-3191
Author(s):  
Johnny Amer ◽  
Zipora Etzion ◽  
Robert M. Bookchin ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Oxidant Stress ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Normal Blood ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Before And After ◽  
Mercury Orange

Abstract Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS2000;97: 8050; BLOOD2000;96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry2004;60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ<1.091 g/ml) RBCs recovered from the interphase layer. The percent val-res RBC in beta-thalassemic samples (n = 10), was 84-fold higher (4.2 ± 0.4% (mean ± SD), range 2.5 to 6.0%) than in normal samples (0.05 ± 0.06%, range 0.02 to 0.1%) (n =10). To determine the oxidative status of the RBC, the cells were washed with PBS and stained for intracellular contents of ROS and GSH, using 2′-7′-dichlorofluoresein and Mercury Orange, respectively. RBC were analyzed by flow cytometry, using gating based on size and granularity. The Mean Fluorescence Channel (MFC) for each fluorochrome was computed. The results showed that valinomycin treatment, per se, did not affect ROS and GSH contents: MFC of the stained un-fractionated RBC was similar before and after treatment with valinomycin, indicating that large changes in MCHC had little or no effect on these measurements. In addition, the unfractionated RBC had ROS and GSH values comparable to those of the high density (val-sensitive) RBC which were recovered from the pellet of valinomycin-treated RBC following Larex fractionation. Measurements on six normal and six beta-thalassemic blood samples indicated that in each case val-res RBC had higher ROS (3.5-10 fold) and lower GSH (2.5-8 fold) levels than the unfractionated RBC or the val-sensitive RBC of the same sample. Compared with val-res cells from normal blood, thalassemic val-res RBC had higher capacity to produce ROS (1.7-fold) and had a lower GSH level (1.5-fold) compared with normal val-res RBC. These results confirm that, as with SCA, beta-thalassemia blood contains a higher percent of val-res RBC than normal blood. They show, further, that (i) both normal and thalassemic val-res RBC have higher oxidative status than other cells (val-sensitive) in the same sample; and that (ii) thalassemic val-res RBC have higher oxidative status than val-res RBC in normal blood. The present results are consistent with the possibility that oxidative stress may contribute to the generation of val-res RBCs, but do not establish a cause-effect relationship. Further studies will be needed to elucidate the origin and significance of these cells.

Oxidative Stress Induces Changes and Shedding of Membrane Phospholipids from Thalassemic RBCs - A NMR Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1782-1782
Author(s):  
Eitan Fibach ◽  
Inna Freikman ◽  
Johnny Amer ◽  
Jack S. Cohen ◽  
Israel Ringel
Keyword(s):  
Oxidative Stress ◽  
31P Nmr ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Membrane Phospholipids ◽  
Flip Flop ◽  
Nmr Study

Abstract Changes in the membrane phospholipid (PL) asymmetry is one of the hallmarks of apoptosis in nucleated cells. Although mature anucleated RBCs, do not undergo the classical pattern of apoptosis, upon trauma or aging they present changes in the membrane asymmetry. These changes include a flip-flop of phosphatidylserine (PS) from the inner leaflet of the membrane to its outer leaflet. This externalization of PS stimulates RBC phagocytosis by macrophages of the reticulo-endothelial system and their removal from the circulation. Oxidative stress is among the causes of PS externalization on RBCs. In beta-thalassemia and sickle cell disease, although the primary defects are mutations in the globin genes, oxidative stress is thought to mediate part of the damage to the RBCs, and particularly to its membrane, including PS externalization. In the present study, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze normal and beta-thalassemic RBCs in order to study the relationship between their oxidative status and the content and shedding of their PL. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio in thalassemic RBCs, confirming their state of oxidative stress. Using 31P-NMR, we measured the content of various PLs, and found 30±3% more phosphatidylcholine (PC), and unexpectedly, less PS in thalassemic RBCs than in normal RBCs. The PS was increased in thalassemic RBC, but not in normal RBC, by treatment with anti-oxidants (vitamin C and N-acetyl cysteine) and decreased by oxidants (t-butylhydroxyperoxide and H2O2) in normal and thalassemic RBCs. PC showed the opposite behavior, indicating a correlation between PS and PC contents and the oxidative status. Since RBCs with exposed PS have been reported to be more frequent in thalassemic blood than in normal blood, we hypothesized that the decrease in PS is a result of shedding from the external membrane, either as free PS moieties or as part of membrane vesicles. NMR analysis of blood plasma obtained from normal and thalassemic donors indicated a 2.6-fold and 1.8-fold increase in PS and PC, respectively in the latter plasma. In vitro incubation of RBC produced much higher PS in supernatants derived from thalassemic RBCs compared with those of normal RBCs. Anti-oxidants reduced the PS shedding from thalassemic RBCs into their supernatants while oxidants increased the PS shedding by normal RBCs. RBCs are known to shed membranous particles (termed vesicles or microparticles) in vitro and in vivo during their physiologic and pathological senescence. We studied this point by purifying microparticles from plasma and RBC supernatants of normal and thalassemic donors, and measuring the PLs content in their lipophilic extracts by 31P-NMR. We found that the PS content and its proportion out of the total PLs were higher in microparticles purified from thalassemic plasma (0.25±0.04 mM, 19% of the plasma total PS) or RBC supernatants than in normal plasma microparticles (0.045±0.06 mM, 9.5% of the plasma total PS) or supernatants. The results also show that although microparticles are enriched in PS compared to their intact RBCs, the bulk of the shed PS is not associated with microparticles. These results suggest that oxidative stress in RBCs causes them to shed their PS and that the increase in PC levels maybe be a compensating mechanism. The pathological consequences of these phenomena on the survival of RBCs in thalassemia warrants further study.

In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1766-1766
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Ada Goldfarb ◽  
Eliezer Rachmilewitz
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia

Abstract In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia. The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005

Platelets and PMN as Well as RBC of Patients with Sickle Cell Anemia Exhibit Oxidative Stress Which Can Be Corrected by Antioxidants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3575-3575
Author(s):  
Johnny Amer ◽  
Hussam Ghoti ◽  
Eliezer Rachmilewitz ◽  
Koren Ariel ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Sickle Cell ◽  
Clinical Manifestations ◽  
Oxidative Status ◽  
Polymorphonuclear Neutrophils ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Beta Thalassemia ◽  
Mercury Orange

Abstract Sickle cell disease (SCD) is caused by an abnormal hemoglobin (HbS), which results mainly in sickling and hemolysis of RBC. However, the platelets and the polymorphonuclear neutrophils (PMN) are also involved in the pathophysiology of the disease. Similar to the findings in thalassemia, some patients develop thromboembolic phenomena with hypercoagulable state, which is due in part to platelet activation. In addition, a growing body of evidence suggests that WBC, particularly PMN, are abnormal; their number is elevated during painful crises and the severity of the disease increases with their number. Several aspects of the changes in the three blood lineages are thought to result from oxidative stress, which represents the imbalance between enhanced generation of reactive oxygen species (ROS) and a low cellular content of antioxidants such as reduced glutathione (GSH), the major intracellular scavenger of ROS. In SCD, oxidative stress results primarily from the premature precipitation of the unstable HbS and the accumulation of excess iron, a catalyst in the formation of ROS. Accumulation of ROS may result in hemolysis of RBC, activation of platelets and a respiratory burst of PMN. We developed flow cytometry techniques for measuring oxidative-state markers, ROS generation and GSH content, simultaneously in RBC, platelets and PMN. Peripheral blood samples obtained from 15 normal donors, 10 patients with SCD and 5 SCD/beta-thalassemia patients (two with the IVS2-1 mutation and 3 with the IVS1-6 mutation) were studied. Out of the 15 SCD patients, 5 were splenectomized, and all were treated with folic acid, except one who was treated with hydroxyurea. The blood was mixed with 3% gelatin, and following 30 min. incubation the upper layer, containing RBC, platelets and WBC, was collected. ROS and GSH were measured by FACS analysis following staining with 2′, 7′-dichlorofuoresceine (DCF) and mercury orange, respectively. Cells were gated on the basis of size and granularity to include platelets, RBC or PMN, exclusively. The cells in each gate were analyzed for green (DCF) or orange (mercury orange) fluorescence and the Mean Fluorescence Channel (MFC) was calculated. The results showed that ROS production increased by 10 to 30-fold in RBC, platelets and PMN from SCD patients compared with that of their normal counterparts. Concomitantly, the GSH content decreased by 20–50% in the SCD cells. It was possible to modulate the oxidative status of cells from both normal donors and SCD patients: Exposure of the cells to oxidants such as hydrogen peroxide (2 mM), hemin (0.1 mM) or iron (ferric ammonium citrate - 0.1 mM) increased the oxidative status in all cell types, while antioxidants such as N-acetyl cysteine, vitamin C (both at 1 mM) and vitamin E (0.2 mM) significantly decreased the oxidative stress. Our results indicate that similar to the findings in thalassemia, in SCD, RBC and platelets, as well as PMN, are in a state of oxidative stress, which could in part account for the clinical manifestations. Addition of antioxidants, which reduced the ROS and enhanced the GSH content of the cells, could protect against oxidative damage. The flow cytometry techniques we developed may prove useful for studying the effects of various antioxidants and for monitoring the patient’s oxidative status during therapy thereby providing an objective, quantitative evaluation of their efficacy.

Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 421-429 ◽  
Author(s):  
Marco Gabbianelli ◽  
Ornella Morsilli ◽  
Adriana Massa ◽  
Luca Pasquini ◽  
Paolo Cianciulli ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Thalassemia Major ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Kit Ligand ◽  
Globin Chains ◽  
In Vitro Response ◽  
Globin Synthesis

In human β-thalassemia, the imbalance between α- and non–α-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in β-thalassemia produced inconsistent results. Here, we investigated the in vitro response of β-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major β-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of γ-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in β-thalassemia major). These studies indicate that in β-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of γ-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

Study assessing the efficacy of herbal teas on bone health and quality of life in a population with osteopenia: rooibos actions on melatonin and tulsi actions on quality of life.

2021 ◽  
Vol 4 (2) ◽  
pp. 270-298
Author(s):  
Fahima Munmun ◽  
Alyssa Linden ◽  
Hunter Hanlon ◽  
Hannah Enderby ◽  
Paula Witt-Enderby
Keyword(s):  
Quality Of Life ◽  
Oxidative Stress ◽  
Bone Health ◽  
Human Mesenchymal Stem Cell ◽  
Melatonin Receptors ◽  
Cooperative Effort ◽  
Translational Study ◽  
Before And After

The purpose of the OsTea translational study was to assess the efficacy of teas (tulsi, rooibos, oolong) compared to placebo (coriander) on markers of bone health and quality of life (QOL) in those with osteopenia and on human mesenchymal stem cell (hMSC) differentiation into osteoblasts to identify potential mechanisms of action. Following consumption of tea (3 times/day; 90 days), participants collected a urine sample during the night (10pm-6am) and filled in questionnaires before and after the study. Rooibos consumption demonstrated a significant decrease in urinary CTX levels vs placebo; trended towards increases in nocturnal melatonin levels (p=0.06); significantly decreased serotonin-producing microbes in the gut; and demonstrated trends towards improvements (p=0.09) in QUALIOST emotional parameters. Tulsi consumption primarily affected subjective measures, such as significantly improved scores for PSS, STAI-trait anxiety, and osteoporosis/osteopenia-related parameters in the QUALIOST. To further identify potential mechanisms underlying these actions of rooibos on CTX and melatonin (urinary and gut), rooibos and melatonin effects on human osteoblastogenesis were carried out for 21 days under oxidative stress conditions to mimic osteopenia.  Although both rooibos and melatonin protected against oxidative stress-induced loss of osteoblasts in vitro, their underlying mechanisms were different.  Melatonin, like tulsi and oolong, demonstrated the greatest protection against oxidative stress at days 10-11 of exposure, which was due to effects on hMSC viability and through melatonin receptors. Rooibos, on the other hand, demonstrated protection at days 10-11 and 20-21, which was through signaling mechanisms involved in differentiation processes and not on cell viability. These findings suggest that the clinical actions of rooibos on decreasing CTX levels in a population with osteopenia may be through a cooperative effort between melatonin and rooibos by protecting hMSC viability against oxidative stress-induced loss and by promoting osteoblast differentiation, respectively.  This study also supports the use of tulsi for improving quality of life in a population susceptible to osteoporosis.

scholarly journals Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1202-1207 ◽  
Author(s):  
A Oppenheim ◽  
Y Katzir ◽  
E Fibach ◽  
A Goldfarb ◽  
E Rachmilewitz
Keyword(s):  
Peripheral Blood ◽  
Globin Gene ◽  
Genetic Regulation ◽  
Inverse Correlation ◽  
Thalassemia Major ◽  
Gene Activity ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
A Site

Abstract Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3′ to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5′ to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5′ to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta- like globin genes in humans.

scholarly journals Oxidative Status before and after Renal Replacement Therapy: Differences between Conventional High Flux Hemodialysis and on-Line Hemodiafiltration

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2809 ◽  
Author(s):  
José Alberto Navarro-García ◽  
Elena Rodríguez-Sánchez ◽  
Jennifer Aceves-Ripoll ◽  
Judith Abarca-Zabalía ◽  
Andrea Susmozas-Sánchez ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Antioxidant Defense ◽  
Oxidized Ldl ◽  
Oxidative Status ◽  
Dialysis Session ◽  
High Flux ◽  
Oxidative Markers ◽  
Before And After ◽  
Total Antioxidant ◽  
On Line

Hemodialysis patients experience high oxidative stress because of systemic inflammation and depletion of antioxidants. Little is known about the global oxidative status during dialysis or whether it is linked to the type of dialysis. We investigated the oxidative status before (pre-) and after (post-) one dialysis session in patients subjected to high-flux dialysis (HFD) or on-line hemodiafiltration (OL-HDF). We analyzed carbonyls, oxidized LDL (oxLDL), 8-hydroxy-2′-deoxyguanosine, and xanthine oxidase (XOD) activity as oxidative markers, and total antioxidant capacity (TAC), catalase, and superoxide dismutase activities as measures of antioxidant defense. Indices of oxidative damage (OxyScore) and antioxidant defense (AntioxyScore) were computed and combined into a global DialysisOxyScore. Both dialysis modalities cleared all markers (p < 0.01) except carbonyls, which were unchanged, and oxLDL, which increased post-dialysis (p < 0.01). OxyScore increased post-dialysis (p < 0.001), whereas AntioxyScore decreased (p < 0.001). XOD and catalase activities decreased post-dialysis after OL-HDF (p < 0.01), and catalase activity was higher after OL-HDF than after HFD (p < 0.05). TAC decreased in both dialysis modalities (p < 0.01), but remained higher in OL-HDF than in HFD post-dialysis (p < 0.05), resulting in a lower overall DialysisOxyScore (p < 0.05). Thus, patients on OL-HDF maintain higher levels of antioxidant defense, which might balance the elevated oxidative stress during dialysis, although further longitudinal studies are needed.

Oxidative Status of Platelets in Normal and Thalassemic Blood.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3765-3765
Author(s):  
Johnny Amer ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Platelet Activation ◽  
Specific Antigen ◽  
Calcium Ionophore ◽  
Iron Chelator ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Ros Generation ◽  
Continuous Exposure ◽  
Light Scatter

Abstract Thromboembolic complications, possibly involving chronic platelet activation, are an important cause of morbidity and mortality in beta-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), has been suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. Previous investigations demonstrated that ROS profoundly affect platelet function and promote platelet activation. Other studies have shown that platelets themselves produce ROS upon activation. In the present study, we adapted flow cytometric techniques to measure oxidative-state markers, ROS generation and reduced glutathione (GSH), using 2′-7′-dichlorofluorescin diacetate and mercury orange, respectively, in platelets. GSH is the major intracellular antioxidant - an important scavenger of ROS. To exclude non-platelets from analysis, a two-parameter (side light scatter and forward light scatter) gate was set. The identity of the gated cells was verified by immunofluorescence staining for CD41 - a platelet-specific antigen. Using these techniques, the average Mean Fluorescence Channel (MFC) values of platelets from 46 normal donors and 22 beta-thalassemic donors were 176 ± 99 vs. 314 ± 81, respectively, for ROS and 319 ± 87 vs. 113 ± 47, respectively, for GSH. These results show that thalassemic platelets contain higher ROS and lower GSH levels than do normal platelets, indicating a state of oxidative stress. The relationship between platelet activation and oxidative status was determined by treating platelets with thrombin (0.1 U/ml), calcium ionophore (5 μM) or phorbol myristate acetate (400 ng/ml). All these treatments caused platelet activation as well as ROS generation; thalassemic platelets were more responsive than platelets from normal controls. In the absence of any known inherent abnormality in thalassemic platelets, the increased oxidative status was attributable to continuous exposure to oxidative insults from extra-platelet sources. Indeed, further investigation indicated that the oxidative status of normal platelets was increased by thalassemic plasma and was inhibited by the iron-chelator Desferoxamin. Iron and hemin, whose levels are increased in thalassemic plasma, stimulated the platelets’ oxidative stress. This was also affected by RBC: it was higher in normal platelets incubated with thalassemic RBC than when incubated with normal RBC. Normal RBC stimulated with hydrogen peroxide, a treatment which results in an elevated oxidative status, increased platelet ROS to a greater extent (3.3-fold) than did unstimulated RBC. These results suggest that thalassemic RBC, having higher than normal ROS, mediate oxidative stress in platelets directly, probably by contact or close proximity. Platelet oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Treatment with these agents of oxidant-stimulated platelets reduced ROS and enhanced the GSH level. The present results indicate that in thalassemia, platelets are in a state of oxidative stress, causing their chronic activation and possibly thromboembolic consequences. This situation may also prevail in other RBC anomalies, such as sickle cell anemia, Polycythemia Vera and Paroxysmal Nocturnal Hemoglobinuria, which are also associated with thromboembolic phenomena. Our findings raise the possibility of using antioxidants in addition to antithrombotic drugs as prophylactic treatment in these diseases.

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