Storage of Erythrocytes Induces Suicidal Erythrocyte Death

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Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca²⁺-activity ([Ca²⁺]_i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca²⁺]_i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca²⁺]_i, increase of ceramide abundance and caspase activation.

“…Moreover, eryptosis is enhanced in elderly individuals [64]. It is sensitive to erythrocyte age [65] and enhanced eryptosis is observed following erythrocyte storage [66, 67]. …”

Section: Introductionmentioning

confidence: 99%

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

Abed

Thiel

Towhid

et al. 2017

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“…Augmented eryptosis is observed in several clinical conditions including iron deficiency [50], dehydration [116], hyperphosphatemia [117], chronic kidney disease (CKD) [118-121], hemolytic-uremic syndrome [122], diabetes [123], hepatic failure [57], malignancy [124, 125], arteriitis [126], sepsis [127], sickle-cell disease [50], beta-thalassemia [50], Hb-C and G6PD-deficiency [50], Wilsons disease [127], as well as advanced age [128]. Eryptosis further increases during storage of blood for transfusion [129]. …”

Section: Introductionmentioning

confidence: 99%

Triggering of Suicidal Erythrocyte Death by Gefitinib

Bhuyan

Wagner

Cao

et al. 2017

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Background/Aims: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca²⁺ entry and/or oxidative stress. Methods: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺. Gefitinib further significantly increased DCFDA fluorescence. Conclusions: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca²⁺ and paralleled by oxidative stress.

“…Eryptosis is stimulated by a myriad of xenobiotics [22, and enhanced eryptosis is observed in a variety of clinical conditions including iron deficiency [22], dehydration [84], hyperphosphatemia [74], vitamin D excess [38], chronic kidney disease (CKD) [85][86][87][88][89] hemolytic-uremic syndrome [90], diabetes [91], hepatic failure [92,93], malignancy [94,95], arteritis [96], sepsis [97], sickle-cell disease [22], beta-thalassemia [22], Hb-C and G6PD-deficiency [22], Wilsons disease [98], as well as advanced age [99]. Eryptosis further increases following erythrocyte storage for transfusion [100].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Suicidal Erythrocyte Death by Volasertib

Bhuyan

Haque

Sahu

et al. 2017

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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 Ca 2+ activity ([Ca 2+ ] 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 Ca 2+ 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-Vbinding, cell volume from forward scatter, [Ca 2+ ] 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