Evolution of structural changes leading to haemolysis in human erythrocytes treated with SK&F 95018, an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties

Abstract

SK&F 95018 is an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties, which, when given to dogs by intravenous infusion, rapidly produced symptoms of intravascular haemolysis. The haemolytic potency of SK&F 95018 was confirmed in vitro using human erythrocytes, was concentration dependent and was associated with dose-specific morphological changes as determined by scanning and transmission electron microscopy. Treatment of washed human erythrocytes with 0.5 m m-SK&F 95018 for up to 30 min resulted in gradual transformation from the biconcave discocyte to stomatocyte forms. Stomatocytes developed more rapidly on exposure to 2 m m-SK&F 95018, exhibited unilateral, multifocal invaginations by 2 min and evolved into spherocytic erythrocytes showing many membrane protuberances and invaginations. At the highest treatment level (10 m m) the crenated erythrocytes seen at time 0 transformed rapidly into spherocytes with many membrane-bound, surface projections that were retained in erythrocyte membrane ‘ghosts’. The membrane-active properties of SK&F 95018 were investigated in a phospholipid-membrane model (an aqueous dispersion of side-chain perdeuterated dipalmitoylphosphatidylcholine) by proton and deuterium nuclear magnetic resonance spectroscopy. The results suggest that SK&F 95018, with its molecular dual polarity, inserts into and effectively disrupts the intergrity of biological membranes by micellar reorganization of the bilayer plasmalemma. The slow change in shape from discocyte to stomatospherocyte at the lowest concentration (without the development of membrane-associated protuberances) suggests a disruptive effect on the erythrocyte osmotic balance by gradual cumulative drug insertion into the membrane. At higher concentrations this initial effect (leading to cell swelling) appears to proceed contemporaneously with micellar membrane reordering, producing membrane protuberances.