scholarly journals Inhibition by Teriflunomide of Erythrocyte Cell Membrane Scrambling Following Energy Depletion, Oxidative Stress and Ionomycin

2016 ◽  
Vol 39 (5) ◽  
pp. 1877-1890 ◽  
Author(s):  
Jens Zierle ◽  
Rosi Bissinger ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Pyrimidine Synthesis ◽  
Tert Butylhydroperoxide ◽  
Stress Energy ◽  
Erythrocyte Surface ◽  
Suicidal Death

Background/Aims: Teriflunomide, an inhibitor of pyrimidine synthesis and thus proliferation of activated T and B lymphocytes, is successfully used for treatment of inflammatory disease. Teriflunomide has further been shown to trigger apoptosis of tumor cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Triggers of cell membrane scrambling include energy depletion, oxidative stress and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether teriflunomide modifies eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, and [Ca2+]i from Fluo3 fluorescence. Results: Oxidative stress (60 min exposure to 0.3 mM tert-butylhydroperoxide), energy depletion (removal of glucose for 48 hours), and exposure to the Ca2+ ionophore ionomycin (1 µM, 60 min) all increased annexin-V-binding, decreased forward scatter and enhanced Fluo3 fluorescence. Teriflunomide (5 µg/ml) did not significantly influence Fluo3 fluorescence, forward scatter and annexin-V-binding under control conditions but significantly blunted the increase of annexin-V-binding following oxidative stress, energy depletion and ionomycin exposure. Teriflunomide further blunted the increase of Fluo3 fluorescence following energy depletion, but did not significantly interfere with increase of Fluo3 fluorescence following oxidative stress and ionomycin exposure. Conclusion: Teriflunomide is a novel inhibitor of suicidal erythrocyte death.

scholarly journals Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment

2016 ◽  
Vol 39 (4) ◽  
pp. 1626-1637 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Stimulation of Erythrocyte Cell Membrane Scrambling by Adarotene

2017 ◽  
Vol 41 (2) ◽  
pp. 519-529 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaàa ◽  
MyriamFezai Fezai ◽  
Mustafa Almasry ◽  
Florian Lang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling ◽  
Ros Formation ◽  
Stimulation Of

Background/Aims: The atypical retinoid E23-(40-hydroxyl-30-adamantylbiphenyl-4-yl) acrylic acid (ST1926, adarotene) is used in the treatment of malignancy. The effect of ST1926 is at least in part due to stimulation of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity [Ca2+]<Sub>i</Sub>, oxidative stress and ceramide. The present study explored, whether adarotene induces eryptosis and, if so, to test for the involvement of Ca2+ entry, oxidative stress and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]<Sub>i</Sub> from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to adarotene (9 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter, as well as significant increase of Fluo3-fluorescence, DCFDA fluorescence, and ceramide abundance. The effect of adarotene (9 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Adarotene stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Triggering of Erythrocyte Cell Membrane Scrambling by Emodin

2016 ◽  
Vol 40 (1-2) ◽  
pp. 91-103 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Microbial Infection ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a component of several Chinese medicinal herbal preparations utilized for more than 2000 years. The substance has been used against diverse disorders including malignancy, inflammation and microbial infection. The substance is effective in part by triggering suicidal death or apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study aimed to test, whether emodin induces eryptosis and, if so, to elucidate underlying cellular mechanisms. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: Exposure of human erythrocytes for 48 hours to emodin (≥ 10 µM) significantly increased the percentage of annexin-V-binding cells, and at higher concentrations (≥ 50 µM) significantly increased forward scatter. Emodin significantly increased Fluo3-fluorescence (≥ 10 µM), DCFDA fluorescence (75 µM) and ceramide abundance (75 µM). The effect of emodin on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Emodin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry and paralleled by oxidative stress and ceramide appearance at the erythroctye surface.

scholarly journals Inhibition of Suicidal Erythrocyte Death by Volasertib

2017 ◽  
Vol 43 (4) ◽  
pp. 1472-1486 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
A.K.M. Ashiqul Haque ◽  
Itishri Sahu ◽  
Hang Cao ◽  
Michael S.D. Kormann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Cell Surface ◽  
Annexin V ◽  
K562 Cells ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Hyperosmotic Shock ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

scholarly journals Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide

2018 ◽  
Vol 50 (6) ◽  
pp. 2283-2295 ◽  
Author(s):  
Madeline Fink ◽  
Abdulla Al Mamun Bhuyan ◽  
Nefeli Zacharopoulou ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation ◽  
Binding Cell

Background/Aims: The sesquiterpene lactone Costunolide is effective against various disorders including inflammation and malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Mechanisms involved include altered function of transcription factors and mitochondria. Erythrocytes lack nuclei and mitochondria but are – in analogy to apoptosis of nucleated cells – able to enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Costunolide induces eryptosis and, if so, to shed light on the mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2’,7’-dichlorodihydrofluorescein (DCF)-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Costunolide (15 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence, DCF-fluorescence, and ceramide abundance. The effect of Costunolide on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Conclusion: Costunolide triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by oxidative stress and ceramide formation.

scholarly journals Inhibition of Erythrocyte Cell Membrane Scrambling Following Energy Depletion and Hyperosmotic Shock by Alectinib

2018 ◽  
Vol 51 (5) ◽  
pp. 1996-2009 ◽  
Author(s):  
Abdulla  Al Mamun Bhuyan ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Glucose Deprivation ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Hyperosmotic Shock ◽  
Anaplastic Lymphoma ◽  
Suicidal Death ◽  
Alk Positive

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor alectinib is clinically used for the treatment of ALK positive non-small-cell lung cancer. At least in part the substance is effective by triggering suicidal death or apoptosis of tumor cells. Erythrocytes are lacking mitochondria and nuclei, key organelles of apoptosis but are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress, and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether alectinib influences eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), hyperosmotic shock (+550mM sucrose for 6 hours), oxidative stress (50 min exposure to 0.3 mM tert-butylhydroperoxide), and Ca2+ loading (60 minutes treatment with 1 µM Ca2+ ionophore ionomycin). Results: A 48 hours exposure of human erythrocytes to alectinib (150-600 ng/ml) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, hyperosmotic shock, oxidative stress and Ca2+ loading were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of energy depletion and hyperosmotic shock, but not of oxidative stress or Ca2+ loading on annexin-V-binding were significantly blunted in the presence of alectinib (150-600 ng/ml). In none of the conditions was forward scatter significantly modified by alectinib. Conclusion: Alectinib inhibits cell membrane scrambling following energy depletion and hyperosmotic shock.

scholarly journals Enhanced Eryptosis Following Auranofin Exposure

2015 ◽  
Vol 37 (3) ◽  
pp. 1018-1028 ◽  
Author(s):  
Kousi Alzoubi ◽  
Jasmin Egler ◽  
Majed Abed ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Methods: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. Results: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). Conclusions: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

scholarly journals Zopolrestat Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (4) ◽  
pp. 1537-1546 ◽  
Author(s):  
Ghada Bouguerra ◽  
Rosi Bissinger ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Binding Cell

Background/Aims: The aldose reductase inhibitor zopolrestat has been shown to either decrease or increase apoptosis, the suicidal death of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how zopolrestat induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, oxidative stress from DCFDA dependent fluorescence, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to zopolrestat (≥ 150 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 125 µg/ml), significantly increased Fluo3-fluorescence (200 µg/ml), significantly increased ceramide abundance (150 µg/ml), but did not significantly modify DCFDA fluorescence. The effect of zopolrestat on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Exposure of human erythrocytes to zopolrestat triggers cell shrinkage and cell membrane scrambling, an effect in part due to Ca2+ entry and ceramide.

scholarly journals Triggering of Suicidal Erythrocyte Death by Fascaplysin

2016 ◽  
Vol 39 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Indole Alkaloid ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation

Background/Aims: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from the hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling

2016 ◽  
Vol 38 (4) ◽  
pp. 1425-1434 ◽  
Author(s):  
Elena Signoretto ◽  
Michela Castagna ◽  
Abdulla Al Mamun Bhuyan ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Human Erythrocyte ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.

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