Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/β‐thalassaemic patients*

Atichartakarn, Vichai; Angchaisuksiri, Pantep; Aryurachai, Katcharin; Onpun, Supornchai; Chuncharunee, Suporn; Thakkinstian, Ammarin; Atamasirikul, Kanlayanee

doi:10.1046/j.1365-2141.2002.03711.x

Cited by 121 publications

(102 citation statements)

References 20 publications

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“…To ascertain the orientation of these extruding vesicles, cultured reticulocytes were stained for PS and with mAb to intracellular epitopes BRIC163 17 (GPA) and BRIC155 18 (AE1), and shown to be inside-out ( Figure 1D). To control for correct PS staining, we treated red cells with N-ethyl maleimide, followed by Ca 21 and ionomycin as Kuypers et al 13 (supplemental Figure 1). We have tested in vitro-produced reticulocytes from numerous different cultures and always observe that 5% to 10% of reticulocytes have an external PS-positive inside-out vesicle.…”

Section: Resultsmentioning

confidence: 99%

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Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

Mankelow

Griffiths

Trompeter

et al. 2015

Blood

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Key Points• Reticulocyte maturation involves the release of intact, inside-out autophagic vesicles with PS exposed on their surface.• Elevated levels of autophagic vesicles on circulating reticulocytes cause PS exposure in patients with SCD.During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 mm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia. (Blood. 2015;126(15):1831-1834) IntroductionThe erythrocyte is one of the most abundant, accessible, and best characterized of human cells but until the recent development of in vitro erythroid culture systems 1,2 obtaining large numbers of its precursor cell, the human reticulocyte has been problematic. Reticulocytes are broadly grouped into R1, motile multilobular, and normally confined to bone marrow, and R2, which are nonmotile, much more mechanically stable, and released into peripheral circulation where they comprise ;2% of red blood cells.3,4 During maturation to an erythrocyte, the reticulocyte must lose ;20% of its surface area, reduce its volume, and degrade or eliminate residual cytosolic organelles. Current dogma considers that loss of plasma membrane is through the release of endocytosed plasma membrane as exosomes, whereas purging of cellular organelles is executed by autophagy. 5Surface phosphatidylserine (PS) exposure is a well characterized signal for initiating phagocytosis of unwanted cells or cellular material. 6PS is normally located on the intracellular surface of plasma membranes. Relocation to the extracellular surface may occur by activation of a scramblase 7 or a bidirectional trafficking process involving cytosolic vesicles.8 PS-exposed red cells are found in the peripheral blood of patients who have undergone splenectomy, or have sickle cell disease (SCD) or thalassemia. 9-13 Study designFor details of anonymized patient samples, antibodies used, and full methods, see supplemental Methods, available on the Blood Web site. Briefly, reticulocytes were derived from erythroid cultures and confocal microscopy was performed as described.2 PS was detected using Annexin V fluore...

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Section: Resultsmentioning

confidence: 99%

“…20 An increased number of PS-exposed red cells has also been observed by flow cytometry in circulating red cells after splenectomy. 21 We analyzed red cells from two splenectomized but otherwise healthy individuals. In both cases, splenectomy was performed because of immune-mediated thrombocytopenia and the patients' hematologic For personal use only.…”

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confidence: 99%

Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

Mankelow

Griffiths

Trompeter

et al. 2015

Blood

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“…12 Circulating MP can be very noxious in different clinical settings such as acute coronary syndromes, atherosclerosis, post-transfusional acute respiratory distress syndrome, diabetes mellitus, systemic inflammatory disease and disseminated intravascular coagulation. 10,12,36,39,40 MP originating from RBC membranes have also been considered a major cause of premature atherosclerosis described in TI patients. 13 The mechanisms responsible for the generation of MP appear very complex in different cell types 32 and very little is known about thalassemic RBC.…”

Section: Discussionmentioning

confidence: 99%

“…10,12,36,39,40 MP originating from RBC membranes have also been considered a major cause of premature atherosclerosis described in TI patients. 13 The mechanisms responsible for the generation of MP appear very complex in different cell types 32 and very little is known about thalassemic RBC.…”

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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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“…It is proven that thalassemic red blood cells have procoagulant activity [15]. Furthermore, a case study done on a splenectomised haemoglobin E/β thalassemia woman with pulmonary arterial hypertension showed normalization of thrombin-antithrombin level and reduce of pulmonary vascular resistance occur after chronic transfusion of packed red blood cells was given in addition to other ongoing treatment with warfarin, acetyl salicylic acid, desferrioxamine, and other supportive measures [16].…”

Section: Discussionmentioning

confidence: 99%

The Level of Natural Anticoagulants in Transfusion Dependent Thalassemia Patients in Kelantan, Northeastern Malaysia

Rosnah

Mn²,

Shafini³

et al. 2014

J Hematol Thrombo Dis

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The association of the severe form of thalassemia with complications of blood transfusion, iron overload, bony deformity and gall stone is well described. Currently, due to improvement in the way of managing thalassemia, most of thalassemia patients survive longer. However more new complications had been observed.Increased incidence of thromboembolism among thalassemia patients had triggered various studies done to determine hypercoagulable state among thalassemia patients. Several factors for the hypercoagulable state had been identified such as RBC membrane disruption, chronic platelet activation and defect in coagulation pathway. This study was carried out to determine the level of Protein C, protein S, free protein S and antithrombin among thalassemia patients and to compare the level of Protein C, protein S, free Protein S and antithrombin level between thalassemia patients and healthy control. This was a case control study done at Hospital Universiti Sains Malaysia. Thirty six thalassemia patients who came for regular blood transfusion and 20 healthy blood donors for normal control were recruited. Blood samples were collected and analyzed for Protein C, protein S, free protein S and antithrombin using ACL Elite Pro. The result showed mean Protein C and free protein S levels were significantly lower (54.8 ± 13.2% and 70.0 ± 12.1% respectively) in thalassemia patients compared to age-matched normal control (94.1 ± 16.3% and 99.8 ± 17.3% respectively), whereas mean total protein S (54.8 ± 13.2% and 94.1 ± 16.3%; respectively) and antithrombin (70.0 ± 12.1% and 99.8 ± 17.3%; respectively) level were similar. In conclusion there was a significantly decreased Protein C and free protein S in thalassemia patients which might suggests hypercoagulable state in thalassemia patients. Since there are a lot of similarities in finding from other studies, we believe that many more study to look for other parameters contributing to hypercoagulable state in thalassemia patients is needed.

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Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/β‐thalassaemic patients*

Cited by 121 publications

References 20 publications

Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease

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

The Level of Natural Anticoagulants in Transfusion Dependent Thalassemia Patients in Kelantan, Northeastern Malaysia

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