Programmed Erythrocyte Death Following in Vitro Treosulfan Treatment

Peter, Thomas; Bissinger, Rosi; Enkel, Sigrid; Alzoubi, Kousi; Oswald, Gergely; Läng, Florian

doi:10.1159/000373958

Cited by 52 publications

(9 citation statements)

References 76 publications

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“…These erythrocytes change into spherocytes by a substantial loss of membrane surface through vesiculation and MPs generation. All these results reveal that IS and IAA trigger a suicidal erythrocyte death that is eryptosis [ 21 , 22 ].…”

Section: Resultsmentioning

confidence: 99%

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Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

Gao

Dong

et al. 2015

Toxins

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Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

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Section: Resultsmentioning

confidence: 99%

“…Procoagulant activity (PCA) of RBCs and MPs was evaluated by one-stage recalcification time assay in a KC4A-coagulometer (Amelung, Labcon, Heppenheim, Germany) [ 21 ]. 100 μL of RBC (1 × 10 8 ) or MPs-containing suspension (prepared from 10 mL of the RBCs supernatants) was incubated with 100 μL of MDP at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

Gao

Dong

et al. 2015

Toxins

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“…Translocation of PS from the inner to the outer membrane leaflet of RBCs is a typical sign of eryptosis (a term introduced by Lang [24]), defining the suicidal death of RBCs. A large variety of physiological parameters as well as substances have been described to induce eryptosis (e.g., [25][26][27][28][29][30][31]). …”

Section: Introductionmentioning

confidence: 99%

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Wesseling

Wagner-Britz²,

Boukhdoud³

et al. 2016

Cell Physiol Biochem

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Background/Aims: The increase of the intracellular Ca²⁺ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca²⁺ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca²⁺ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca²⁺ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca²⁺ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca²⁺ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

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“…Signaling contributing to stimulation of eryptosis includes activated caspases, stimulated activity of casein kinase 1 α , Janus‐activated kinase JAK3, protein kinase C, and p38 kinase, as well as impaired activity of AMP activated kinase AMPK, cGMP‐dependent protein kinase, mitogen‐activated kinase and stress‐activated kinase MSK, PAK2 kinase, and sorafenib/sunitinib sensitive kinases . Eryptosis is triggered by a wide variety of xenobiotics and enhanced eryptosis is observed in several clinical conditions including dehydration, hyperphosphatemia, chronic kidney disease (CKD), hemolytic‐uremic syndrome, diabetes, hepatic failure, malignancy, sepsis, sickle‐cell disease, beta‐thalassemia, Hb‐C and G6PD‐deficiency, as well as Wilsons disease …”

Section: Introductionmentioning

confidence: 99%

Ceranib‐2‐induced suicidal erythrocyte death

Signoretto

Zierle

Bhuyan

et al. 2016

Cell Biochemistry & Function

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Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca(2+) -activity ([Ca(2+) ]i ) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib-2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine-exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib-2 significantly increased the percentage of annexin-V-binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib-2 significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib-2 on annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) . Ceranib-2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca(2+) entry. Copyright © 2016 John Wiley & Sons, Ltd.

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Programmed Erythrocyte Death Following in Vitro Treosulfan Treatment

Cited by 52 publications

References 76 publications

Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Ceranib‐2‐induced suicidal erythrocyte death

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