Red Cell Shape

By M. A. LICHTMAN and G. V. MARINETTI. ERYTHROCYTES in plasma at 37° C change from disc to sphere when ATP falls to <0.25 flmoles/ml RBC at -16 hours. From 0 to 16 hours, intracellular calcium was zero but rose to 0.11 and 0.51 X 10-17 moles,! cell at 24 and 48 hours. Membrane calcium (0.14 X 10-17 moles/cell) remained unchanged over 48 hours. Erythrocytes in autologous plasma at 37° C were nearly all (>85 %) crenated spheres after 18 hours (ATP <0.15 flmoles/ml) and were tightly crenated or smooth spheres after 36 hours (ATP <0.08 moles/mI). However, erythrocytes were largely (>75 %) discs at 18 hours and many were discoidal from 18 to 36 hours if examined in fresh plasma indicating a plasma factor in spherogenicity although injury does become irreversible. Plasma incubated at 37° C could by -10 hours convert autologous fresh erythrocytes to crenated spheres (echinocytes) [1 J. Plasma echinocytogenic capacity was accompanied by a conversion of lecithin to lysolecithin and cholesterol to cholesterol-ester (plasma lecithin: cholesterol acyl-transferase (LCAT). Lysolecithin added to plasma in similar amounts produced morphologically identical echinocytes. Incubated whole blood contained in its plasma echinocytogenic capacity for fresh autologous erythrocytes at a time when erythrocytes present in whole blood were not echinocytes (-10 hours). Spherocytosis did not ensue until ATP fell to <.25 flmoles/ml. Moreover, erythrocytes added to preincubated (24 hour) plasma and instantaneously converted to echinocytes, returned to discs after 6 hours further incubation in the echinocytogenic plasma; and, discs persisted if erythrocyte ATP remained >0.25 I-tmoles/ml. Endogenous or exogenous increases in lysolecithin resulted in small aggregates of erythrocytes which persisted when echinocytes reverted to discs by further incubation (?cell fusion). Red cells were added to echinocytogenic (24 hour 36 M. A. LICHTMAN AND G. V. MARINE TTl preincubated) plasma containing 1,3-14C-Iysolecithin and membrane preparations prepared immediately and at selected intervals over 24 hours. Lysolecithin incorporated into the red cell membrane rapidly. The resumption of the disc shape of most cell over 6 hours occurred without a reduction in the membrane content of HC-Iysolecithin. Only later did membrane HC-Iysolecithin fall and HC-Iecithin increase (acylation). Therefore, the resumption of the disc shape cannot be accounted for by acylation of lysolecithin, rather other processes, perhaps involving lipid exchange, makes it possible for the red cell to resume a near discoidal shape despite the presence of large (echinocytogenic) concentrations of lysolecithin remaining in plasma and membrane. This protective process must also occur when whole blood is incubated in vitro since lysolecithin formation occurs in the plasma of whole blood, yet the red cells in whole blood incubations do not undergo echinocytosis at a time when plasma has echinocytogenic capacity for freshly added autologous red cells. Echinocytosis occurs again, at a later time, when ATP falls. Hence, the membrane lipid changes of red cells observed during the initial hours of incubation in vitro before severe ATP depletion may be related to compensation for lysolecithin accumulation (LCAT activity). Later membrane changes in red cells are correlated with and subsequent to ATP depletion and cellular calcium accumulation [2J. The generation of lysolecithin by LCAT does not occur at refrigerator (4 0 C) temperature over 6 weeks and hence is not consequential as a factor in cell injury during blood storage. (University of Rochester. School of Medicine and Dentistry, Rochester, New York, U.S.A.)