Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

Westerman, Maxwell P.; Pizzey, Arnold; Hirschman, Jocelyn; Cerino, Mario; Weil-Weiner, Yonit; Ramotar, Prya; Eze, Ada; Lawrie, Andrew; Purdy, G.; Mackie, Ian; Porter, John B.

doi:10.1111/j.1365-2141.2008.07155.x

Cited by 143 publications

(166 citation statements)

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“…It has been reported that splenectomy in thalassemia intermedia patients is associated with higher plasma hemoglobin and red cell microparticle levels. 42 There are a number of limitations to this study. Episodes of painful vaso-occlusive crisis, acute chest syndrome and priapism were self-reported and, therefore, subject to recall bias.…”

Section: Discussionmentioning

confidence: 95%

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

et al. 2012

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464haematologica | 2013; 98(3)The intensity of hemolytic anemia has been proposed as an independent risk factor for the development of certain clinical complications of sickle cell disease, such as pulmonary hypertension, hypoxemia and cutaneous leg ulceration. A composite variable derived from several individual markers of hemolysis could facilitate studies of the underlying mechanisms of hemolysis. In this study, we assessed the association of hemolysis with outcomes in sickle cell anemia. A hemolytic component was calculated by principal component analysis from reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and total bilirubin concentrations in 415 hemoglobin SS patients. Association of this component with direct markers of hemolysis and clinical outcomes was assessed. As primary validation, both plasma red blood cell microparticles and cell-free hemoglobin concentration were higher in the highest hemolytic component quartile compared to the lowest quartile (P≤0.0001 for both analyses). The hemolytic component was lower with hydroxyurea therapy, higher hemoglobin F, and alpha-thalassemia (P≤0.0005); it was higher with higher systemic pulse pressure, lower oxygen saturation, and greater values for tricuspid regurgitation velocity, left ventricular diastolic dimension and left ventricular mass (all P<0.0001). Two-year follow-up analysis showed that a high hemolytic component was associated with an increased risk of death (hazard ratio, HR 3.44; 95% confidence interval, CI: 1.2-9.5; P=0.02). The hemolytic component reflects direct markers of intravascular hemolysis in patients with sickle cell disease and allows for adjusted analysis of associations between hemolytic severity and clinical outcomes. These results confirm associations between hemolytic rate and pulse pressure, oxygen saturation, increases in Doppler-estimated pulmonary systolic pressures and mortality (Clinicaltrials.gov identifier: NCT00492531). The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

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Section: Discussionmentioning

confidence: 95%

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

et al. 2012

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“…Indeed splenectomized patients with thalassemia intermedia have significantly higher levels of microparticles and thrombin-antithrombin complexes when compared to non-splenectomized patients. 39 Furthermore, the levels of phosphatidylserine-positive microparticles and plasma hemoglobin were higher in the splenectomized patients, although this difference did not reach statistical significance. 39 The utility of anticoagulation in hemolytic anemias prior to the development of thrombotic complications is uncertain.…”

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confidence: 88%

“…39 Furthermore, the levels of phosphatidylserine-positive microparticles and plasma hemoglobin were higher in the splenectomized patients, although this difference did not reach statistical significance. 39 The utility of anticoagulation in hemolytic anemias prior to the development of thrombotic complications is uncertain. While it is reasonable to use prophylactic anticoagulants to decrease the risk of deep vein thromboses when these patients are hospitalized, there is a paucity of controlled trials in this setting.…”

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confidence: 88%

Hypercoagulability and thrombotic complications in hemolytic anemias

Ataga¹

2009

Haematologica

150

114

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“…7,8 Splenectomy, performed to alleviate anemia in TI patients, may result in severe thrombotic episodes 9,10 and may cause a rise of pro-thrombotic circulating microparticles (MP). 11 The composition and pathogenic roles of MP have been extensively studied in various diseases, such as ischemia, diabetes and atherosclerosis, revealing complex pathogenic roles in modulating nitric oxide and prostacyclin production, stimulating cytokine release, inducing tissue factor expression, as well as monocyte chemotaxis and adherence to the endothelium. [12][13][14][15][16][17][18] Recent studies on the mechanisms of redox regulation of RBC membrane stability 19,20 indicate that oxidative stress induces a phosphorylative response that specifically involves two tyrosine residues located in the cytoplasmic domain of band 3.…”

Section: Introductionmentioning

confidence: 99%

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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Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy

Cited by 143 publications

References 31 publications

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

Hypercoagulability and thrombotic complications in hemolytic anemias

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

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