Clofazimine Induced Suicidal Death of Human Erythrocytes

Abstract

Background/Aims: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A₂ and to induce apoptosis. In erythrocytes phospholipase A₂ stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A₂ is in part effective by fostering formation of prostaglandin E₂, which triggers Ca²⁺ entry. Stimulators of Ca²⁺ entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca²⁺ activity ([Ca²⁺]_i) from Fluo3-fluorescence, reactive oxygen species (ROS) from 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. Results: A 24-48 hours exposure of human erythrocytes to clofazimine (≥1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca²⁺]_i, significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca²⁺, and by phospholipase A₂ inhibitor quinacrine (25 µM). Clofazimine further augmented the effect of Ca²⁺ ionophore ionomycin (0.1 µM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca²⁺ removal and addition of quinacrine. Conclusion: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca²⁺, paralleled by cellular energy depletion and sensitive to phospholipase A₂ inhibitor quinacrine.

Ceranib‐2‐induced suicidal erythrocyte death

“…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 …”

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin

“…It is, therefore, tempting to speculate that pyocyanin‐induced eryptosis may participate in the thrombogenic complications of P. aeruginosa sepsis . In addition to septicaemia, eryptosis participates in the pathophysiology of a wide range of systemic conditions and is associated with the toxicity of various biologically active compounds . Furthermore, eryptosis is sensitive to erythrocyte age and is an important determinant of the quality of stored erythrocytes for transfusion .…”

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

“…Further signaling molecules that regulate the eryptosis machinery include AMPK 20 , p38 MAPK 31 , CK1α 32 , PAK2 33 , PDK1 20 , MSK1/2 34 and CDK4 35 . Eryptosis is triggered by a myriad of xenobiotics and endogenous substances 20 36 37 38 39 40 41 42 43 44 45 46 47 48 , and accelerated eryptosis contributes to the anemia associated with several clinical disorders 20 , including iron deficiency 49 , sepsis 50 , renal failure 51 , hepatic failure 52 , malignancy 24 , ageing 53 and Wilson’s disease 54 . Eryptotic erythrocytes adhere to the vascular wall 55 , and stimulate blood clotting 56 .…”