Electric field pulses induce reversible shape transformation of human erythrocytes

Abstract

Electric field pulses >2-3 kV cm¹ long known to induce membrane poration and fusion of erythrocytes as well as to enhance the transbilayer mobility of phospholipids and to perturb aminophospholipid asymmetry, are shown to induce, at 0 C., transformation of the discocytic cells into echinocytes and spheroechinocytes. The extent of transformation increases with strength, duration and number of pulses. Its time course is biphasic., a major rapid phase (t/2 ∼ 5 s) being followed by a minor one, lasting for 2-3 h. Shape transformation goes along with the exofacial exposure of phosphatidylserine (PS), detected by FITC-annexin V binding and quantified by a calibration curve established via externally inserted dilauroylphosphatldylserine. Incubation of these echinocytes at 37 C leads to a rapid recovery of the discocytic shape followed by slower formation of stomatocytes. Shape recovery is temperature dependent (E_a ∼100 kJ/mol), and can be impaired by depletion of ATP or Mg⁺⁺ and by addition of vanadate or fluoride. Shape recovery and stomatocyte formation go along with a rapid loss of annexin binding in about 45% of the cells while the rest maintains its binding capacity. In the presence of vanadate, annexin binding increases in all cells. The results are discussed in the light of the bilayer couple concept of erythrocyte shape and the enhanced transverse mobility of phospholipids. Echinocyte formation is most likely caused by the reorientation of endofacial aminopho-spholipids to the outer leaflet of the bilayer. Shape recovery and stomatocyte formation probably result from a continuous reinternalization of PS via the ATP dependent aminophospholipid translocase, but may also be supported by downhill movement of PC to the inner leaflet and by other yet unidentified processes.

Key Words:

• erythrocyte membrane
• electroporation
• erythrocyte shape
• aminophospholipid translocase
• annexin V