After incorporation of spin-labeled phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine analogues in the outer leaflet of the plasma membrane in resting platelets, more than 90% amino-head analogues accumulated within 30 min in the inner leaflet by aminophospholipid translocase activity, while choline analogues mostly remained on the outer leaflet. Platelets were then activated by thrombin or Ca2+ ionophore A23187. No outward movement of internally located spin-labeled aminophospholipids was observed during thrombin-induced activation, whereas the influx of externally located probes increased slightly. During A23187-mediated activation, similar slightly increased influx was observed, while 40-50% of the initially internally located aminophospholipids could then be extracted from the outer leaflet. This sudden exposure on the outer face was dependent on an increase in intracellular Ca2+ and achieved in less than 2 min at 37 degrees C. Inhibition of translocase activity by N-ethylmaleimide did not induce any aminophospholipid outflux. When probes were incorporated on the outer face of the plasma membrane in resting platelets, they were still fully accessible from the extracellular medium after A23187-induced activation. Moreover, they were distributed between the vesicles and remnant platelets in proportion to the external membrane phospholipidic content in each structure. This suggested that no scrambling of plasma membrane leaflets occurred during the vesicle blebbing. Moreover, the spin-labeled aminophospholipids exposure rate and amplitude were unchanged when vesicle formation was inhibited by the calpain inhibitor calpeptin. These results indicate that loss of asymmetry thus inducing generation of a catalytic surface is not the consequence of vesicle formation. Conversely, we propose that vesicle shedding is an effect of PL transverse redistribution and calpain-mediated proteolysis during activation.

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The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short-chain spin-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10 -70 mM benzyl alcohol. A dual effect was observed: (a) at 4°C and 37°C there was an N-ethylmaleimide-sensitive, long lasting and fully reversible increase in the spin-labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37"C, an enhancement of N-ethylmaleimide-insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose-dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose-dependent shape changes : an immediate one from discocytes to echinocytes, followed by a slower N-ethylmaleimide-and ATP-dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37 "C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy-consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37°C.Benzyl alcohol has been observed to modify the activity of numerous membrane proteins [I -41 and more complex mechanisms, for example endocytosis [5]. Its effect on membranous enzyme activity is often related to a change in membrane fluidity, as is the case for other local anaesthetics. Furthermore, it has been postulated that benzyl alcohol has a direct effect on proteins [6]. Inhibition of the fusion stage between lysosomes and endocytic vesicles was also suggested to explain the effect of benzyl alcohol on asialo-glycoprotein receptors [7]. The effect of benzyl alcohol on phospholipid reorientation kinetics and transverse repartition in the red cell membrane is also noteworthy. Transverse distribution of phospholipids in the erythrocyte membrane is asymmetric, mainly regulated by a specific aminophospholipid carrier protein, aminophospholipid translocase [8 -101. Translocase activity in erythrocytes was found to be sensitive to certain membrane perturbations, due to cholesterol/phospholipid ratio variation [ I l l or chlorpromazine incorporation [I 21, for example. Conversely, other amphiphdic drugs do not modify translocase activity [12]. There are several advantages to using benzyl alcohol as a membrane-perturbing agent: (a) this molecule is neutral and does not preferentially invade one of the two membrane leaflets, thus avoiding hemolysis even at a high concentration; (b) being a neutral compound precludes any selective interaction with charg...