Eryptosis: a driver of anemia in chronic kidney disease.

PURPOSE OF REVIEW: Anemia, characterized by a reduction in red blood cell (RBC) count or hemoglobin concentration, commonly accompanies chronic kidney disease (CKD), significantly impacting patients' quality of life. This review delves into the multifaceted nature of anemia in CKD, with a focus on novel mechanisms, particularly the dysregulation of eryptosis or programmed cell death of RBCs, leading to shortened RBC lifespan. RECENT FINDINGS: Recent studies in CKD patients and mouse models revealed that eryptosis, driven by factors such as uremic toxins, inflammation, and imbalances in calcium homeostasis, plays a pivotal role in the development of renal anemia. Dysregulated eryptosis results in premature RBC destruction, exacerbating the hypoproliferative character of anemia in CKD. SUMMARY: Recognizing the intricate relationship between eryptosis and anemia in CKD opens promising prospects for improving patient outcomes and enhancing our understanding of this complex condition. Future research and therapeutic development in this area hold the potential to improve anemia treatment of CKD patients.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism.

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.

BACKGROUND: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Inhibition of Collagen Related Peptide Induced Platelet Activation and Apoptosis by Ceritinib.

BACKGROUND/AIMS: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 - 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. CONCLUSIONS: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

Suicidal death of erythrocytes in cancer and its chemotherapy: A potential target in the treatment of tumor-associated anemia.

In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis characterized by cell shrinkage and cell membrane scrambling. Eryptotic erythrocytes are rapidly cleared from circulating blood and may adhere to the vascular wall. Stimulation of eryptosis thus impairs microcirculation and leads to anemia as soon as the loss of erythrocytes cannot be fully compensated by enhanced erythropoiesis. Signaling stimulating eryptosis includes increase of cytosolic Ca2+ -activity, ceramide, caspases, calpain, p38-kinase, protein-kinase C, Janus-activated kinase 3, casein-kinase 1α, and cyclin-dependent kinase 4. Eryptosis is inhibited by AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein-kinase, mitogen- and stress-activated kinase, and sorafenib- and sunitinib-sensitive tyrosine-kinases. Eryptosis is triggered by complement, hyperosmotic shock, energy-depletion, oxidative stress, multiple xenobiotics including diverse cytostatic drugs, diabetes, hepatic failure, iron-deficiency, chronic kidney disease, hemolytic-uremic-syndrome, fever, systemic lupus erythematosus, infections, sepsis, sickle cell anemia, thalassemia, glucose-6-phosphate-dehydrogenase deficiency, and Wilson´s disease. Compelling evidence points to a decisive role of eryptosis in anemia of malignancy. As shown for lung cancer, eryptosis inducing plasma components accumulate in cancer patients and trigger oxidative stress and ceramide. The tumor-induced eryptosis leads to anemia despite increased erythropoiesis. The stimulation of eryptosis in malignancy is compounded by cytostatic treatment, as a large number of cytostatic agents trigger eryptosis. Inhibiting eryptosis may be a useful strategy in reducing tumor-induced anemia and impaired microcirculation. Inhibitors of eryptosis may, however, be harmful, if they similarly interfere with death of tumor cells. Clearly, additional experimental effort is required to achieve killing of tumor cells with simultaneous avoidance of stimulated eryptosis.

Inhibition of Erythrocyte Cell Membrane Scrambling by ASP3026.

BACKGROUND/AIMS: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis. METHODS: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. RESULTS: Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. CONCLUSIONS: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death.

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

Triggering of Suicidal Erythrocyte Death by Exemestane.

BACKGROUND/AIMS: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, 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, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved. METHODS: 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 (ROS) abundance from DCF fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM). CONCLUSIONS: Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca2+]i, oxidative stress, and increased ceramide abundance.

Temsirolimus Sensitive Stimulation of Platelet Activity, Apoptosis and Aggregation by Collagen Related Peptide.

BACKGROUND/AIMS: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus stimulates apoptosis of tumor cells and is thus therapeutically used for the treatment of diverse malignancies. On the other hand, temsirolimus has been shown to protect against apoptosis of hippocampal neurons. Similar to nucleated cells, blood platelets may enter suicidal death characterized by cell shrinkage and cell membrane scrambling. Platelet apoptosis is frequently preceded by Ca2+ entry, degranulation, integrin activation and stimulation of caspases. Those events could be triggered by collagen related peptide (CRP). The present study explored whether treatment of platelets with temsirolimus modifies platelet activation, caspase activity, platelet shrinkage, and phosphatidylserine abundance. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to temsirolimus (40 µg/ml) without or with additional CRP (2 µg/ ml or 5 µg/ml) treatment. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fuorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding and relative platelet volume from forward scatter. RESULTS: In the absence of CRP, the administration of temsirolimus (40 µg/ml) significantly decreased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, cell volume, caspase activity and aggregation. Exposure of platelets to CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbß3 integrin activity, annexin-V-binding, ROS, caspase activity and aggregation, effects significantly blunted in the presence of temsirolimus. CRP further decreased forward scatter, an effect again significantly blunted by temsirolimus. CONCLUSIONS: Temsirolimus is a powerful inhibitor of platelet activation and suicidal platelet death.

Effect of Bexarotene on Platelet Activation and Apoptosis.

BACKGROUND/AIMS: The retinoid X receptor (RXRs) stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl] benzoic acid) is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i), which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP). The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, and relative platelet volume from forward scatter. RESULTS: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, active αIIbß3 integrin, annexin-V-binding, and caspase activity. The effects of thrombin on [Ca2+]i, annexin-V-binding, cell volume, and caspase activity as well as the effects of CRP on [Ca2+]i, P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, cell volume, and caspase activity were significantly augmented in the presence of bexarotene. CONCLUSIONS: Bexarotene sensitizes blood platelets for thrombin and/or CRP induced activation and apoptosis.

Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation.

BACKGROUND/AIMS: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]_i), oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling. METHODS: Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid), Pam2 (lipopeptide with two fatty acids), or Pam3 (lipopeptide with three fatty acids). In the following phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]_i from Fluo3-fluorescence, ROS formation from DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies. RESULTS: Pam1 (5 µg/ml), Pam2 (5 µg/ml) and Pam3 (1 and 5 µg/ml) significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS) and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis. CONCLUSIONS: Lipopeptides are not only important activators of the immune system; at higher concentrations they also drive host cells into apoptosis thus aggravating a bacterial infection.

Effects of Antimalarial Tafenoquine on Blood Platelet Activity and Survival.

BACKGROUND/AIMS: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+]i, P-selectin abundance, active αIIbß3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+]i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+]i, P-selectin abundance, and active αIIbß3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. CONCLUSIONS: Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation.

Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis.

BACKGROUND/AIMS: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbß3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbß3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. CONCLUSIONS: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

BACKGROUND: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. METHODS: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. RESULTS: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. CONCLUSIONS: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

BACKGROUND/AIMS: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. METHODS: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. RESULTS: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. CONCLUSIONS: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Eryptosis - the Neglected Cause of Anemia in End Stage Renal Disease.

End stage renal disease (ESRD) invariably leads to anemia which has been mainly attributed to compromised release of erythropoietin from the defective kidneys with subsequent impairment of erythropoiesis. However, erythropoietin replacement only partially reverses anemia pointing to the involvement of additional mechanisms. As shown more recently, anemia of ESRD is indeed in large part a result of accelerated erythrocyte loss due to suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface. Phosphatidylserine exposing erythrocytes are bound to and engulfed by macrophages and are thus rapidly cleared from circulating blood. If the loss of erythrocytes cannot be fully compensated by enhanced erythropoiesis, stimulation of eryptosis leads to anemia. Eryptotic erythrocytes may further adhere to the vascular wall and thus impair microcirculation. Stimulators of eryptosis include complement, hyperosmotic shock, energy depletion, oxidative stress, and a wide variety of xenobiotics. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, and cyclin-dependent kinase 4. Eryptosis is inhibited by AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and some illdefined tyrosine kinases. In ESRD eryptosis is stimulated at least in part by a plasma component, as it is triggered by exposure of erythrocytes from healthy individuals to plasma from ESRD patients. Several eryptosis-stimulating uremic toxins have been identified, such as vanadate, acrolein, methylglyoxal, indoxyl sulfate, indole-3-acetic acid and phosphate. Attempts to fully reverse anemia in ESRD with excessive stimulation of erythropoiesis enhances the number of circulating suicidal erythrocytes and bears the risk of interference with micocirculation, At least in theory, anemia in ESRD could preferably be treated with replacement of erythropoietin and additional inhibition of eryptosis thus avoiding eryptosis-induced impairment of microcirculation. A variety of eryptosis inhibitors have been identified, their efficacy in ESRD remains, however, to be shown.

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

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.