Stimulation of ceramide formation and suicidal erythrocyte death by vitamin K3 (menadione)

Vitamin K3 is an essential micronutrient required for the activation of coagulation factors and thus hemostasis. Administration of vitamin K3 analogues may cause anemia, which at least in theory could be due to stimulation of suicidal erythrocyte death or eryptosis characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane leading to exposure of phosphatidylserine at the erythrocyte surface. Eryptosis is triggered by an increase in the cytosolic Ca2+ activity, by ceramide and by energy depletion (decrease of cytosolic ATP). The present experiments explored, whether vitamin K3 may influence eryptosis. Hemolysis was estimated from the supernatant hemoglobin concentration, phosphatidylserine-exposing erythrocytes from annexin V-binding in fluorescence-activated cell sorter (FACS) analysis, erythrocyte volume from forward scatter in FACS analysis, ceramide formation from binding of fluorescent antibodies, and erythrocyte ATP content from a luciferin–luciferase assay. As a result, vitamin K3 (≥1 µM) caused lysis of an only small fraction of erythrocytes, but significantly increased ceramide formation, significantly increased the percentage of annexin V-binding erythrocytes, significantly decreased forward scatter and, at higher concentrations, significantly decreased the cellular ATP content. In conclusion, vitamin K3 stimulates suicidal erythrocyte death, an effect at least partially due to ceramide formation and ATP depletion.