Effect of Drugs on Human Erythrocytes. II. A Possible Mechanism of Drug-induced Hemolysis

Many kinds of drugs cause lysis of erythrocytes at higher concentrations. To clarify the mechanism of hemolysis, the effect of chlorpromazine and clemastine on the structure and arrangement of the membrane proteins and phospholipids was studied using circular dichroism (CD) and some hydrophobic probes, in addition to studies on the alterations in the components of the cells and using "pink ghosts"containing α-amylase. Initial experiments demonstrated that human erythrocytes were hemolyzed within several minutes at a higher drug concentration (1×10-3 or 8×10-4M), while a somewhat lower concentration (6 or 5×10-4M) there was a lag phase for about 30 min and then resulted in severe hemolysis. Dramatic hemolysis occurred at a chlorpromazine concentration which approximately corresponds with the critical micellar concentration. The content of sulfhydryl groups in the cells and calcium in the membrane was not affected by the drug treatment. At above 2×10-4M of both drugs, components I and II (spectrin) were released from the membrane. These drugs, however, had little or no effect on the CD spectra of the ghosts, suggesting that the drug-induced hemolysis is not due to the denaturation of the proteins. 1-Anilinonaphthalene 8-sulfonate (ANS)-ghost fluorescence and 2, 4, 6-trinitrobenzenesulfonate (TNBS) binding to aminophospholipids were extremely enhanced by the drug exposure. The enhancement of the ANS-ghost fluorescence and TNBS binding with drugs was almost paralleled with that of the hemolytic effect. These drugs thus disturb the arrangement of phospholipids and the hydrophobic interactions between lipids and proteins, and alter the membrane permeability, thereby appear to induce hemolysis.