Brief iiteports
Red Blood Cell Membrane Storage Lesion S. L. SCHRIER,B. HARDY,K. BENSCH,I. JUNGAA N D J. KRUEGER From the Departments of Hematology and Pathology. Stanford University School of Medicine, Stanford, California StORep d human erythmcytesin CPD produced a lesion of the erythrocyte membrane manifested by abnormal endocytosb in resealed ghosts and in intact erythrocytes. Endocytosis produced by rescaling Ca, Mg, and ATP into g b t a was impaired by Rve weeks of storage and this defect was promptly reversed by the prior repneratbn of ATP in the stored erytbmcytes. DrugInduced endocytods was studied in intact stored erythrocytes. VinblPstine endocytosis was not affected by storage. Chlorpromazlne endocytmls was variably but never compktely inhibited by storage, and restoration of ATP occasbndy resulted in compkte restoration of chlorpromsdne endocytosis to base line values. However, prlmaquine endocytosis was usually totally inhibited after three to four weeks of storage at a time when residual ATP kvels were 30 to 50 per cent of baae Une values. Restoration of ATP levels to at kast baae line values dM not compktely restore primaquine endocytosls to control values. Study of primaquine endocytosrs provides an opportunity for delning an erythrocyte membrane storage kslon.
A VARIETY of methods have been used to extend the allowable blood bank storage period for erythrocytes. Supplementation of the anticoagulant solution has been extensively studied with the aim of avoiding the metabolic storage lesion which usually means preserving relatively high levels of ATP, and ~ , ~ - D P G . 'Persistence B'~ of ATP correlates imperfectly with the 24 hour posttransfusion survival of erythrocyte^,^.'^ however, the mechanisms by which ATP might work have not been clearly established. Since ATP has known membrane functions, it is possible that ATP depletion induced by storage, could affect erythrocyte membrane functions." Membrane storage lesions have been described. These have Supported by NIH Grant AM 13682. A preliminary report of this work was presented to the American Society of Hematology. Received for publication January 23, 1978; accepted February 27, 1978.
included alterations in membrane polypept i d e ~enhanced ,~ accessability of membrane proteins to external probes,Band increased agglomeration of erythrocytes in media of low ionic strength at pH 7.2.8 Theoretically, if a membrane storage lesion developed, perhaps as a consequence of prolonged ATP depletion, it could ultimately limit the shelf life of erythrocytes stored in liquid media. We therefore proposed to determine if there was a functional erythrocyte membrane storage lesion by measuring endocytosis in vitro in intact erythrocytes' and in their resealed ghosts1* because endocytosis involves a variable interaction between ATP and the membrane functions of invagination and fusion.'J9 Materials and Methods Seven normal donors participated in various aspects of the experiments according to protocols and informed consent forms approved by the Stanford University Committee on Human Experimentation. Venous blood was drawn directly into CPD contained in sterile 16 mm x 125 mm Falcon Plastic Screw-top Tubes using a ratio of 0.14 ml of CPD per ml of whole blood. The anticoagulated blood was stored at 4 C and the tubes were inverted once at weekly intervals. ATP levels in erythrocytes were determined by the coupled hexokinase-glucose-6-phosphate dehydrogenase reaction in neutralized perchloric acid filtrates and reported as p moles per ml of packed RBC. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on "white Dodge ghosts'' by the method of Fairbanks. Endocytosis in intact erythrocytes was induced by incubation with primaquine. chlorpromazine, and vinblastine using the materials and methods previously described.**14 In intact erythrocytes, endocytosis was evaluated by
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MEMBRANE STORAGE LESION
phase microscopy and by trapping of T o Vitamin BI2within endocytic vacuole^.^.^^ Endocytosis in red blood cell ghosts was induced by resealing 2.5 mM Mg-ATP and 1.0 mM 'Va within the ghosts.'* Measurement of red blood cell ghost endocytosis was achieved by phase and transmission electron microscopy (TEM). We also measured ' T a efflux. and the amount of residual 45Ca binding to red ghost membranes." Because there is a degree of variability in measuring quantitative aspects of endocytosis the experiments were usually designed so that blood was withdrawn from the normal donor at stipulated times before the final day on which all the experiments were performed. Therefore, the endocytosis data for a single experiment is derived from measurements made on one day using the same reagents and controls. Restoration of ATP levels was achieved by incubating erythrocytes at 37 C for one hour with the pyruvate-inosine-glucose-phosphate-adenine (PIGPA) medium exactly as described by Valeri.Is
ATP CONTENT OF R E
---
Results .I
ATP Content of Erythrocytes The data for one experiment are shown in Figure 1. Incubation with the PIGPA medium resulted in the restoration of ATP levels at least to base line values.
0
I
EsTmEOBT )IIQIIlTnN 2
s
4
c
=pCo#.
S
6
WEEKS SrORlyT
FIG.1. ATPlevels plotted against storage in weeks (solid line). The dotted line indicates the ATP values after the samples had been incubated for one hour with the PIGPA medium.
Resealed Red Blood Cell Ghosts We had previously reported that in red blood cell ghosts there was a correlation between endocytosis, and activity of Ca, Mg-ATPase and *Ca efflux.'* Therefore, 'Ta efflux was measured in red blood cell ghosts made from stored erythrocytes and no impairment in 45Caeftlux
was observed (Table 1). In sickle cell anemia there is both deficient ghost endocytosis and a five-fold increase above normal in the amount of 45Ca remaining adherent to erythrocyte ghost membranes after 30 minutes of incubation at
Table 1. %a €ff/ux from Resealed Ghosts Length of Storage (weeks)
0
1.5
3
4
5
6
1.39 100
0.89 117
0.68 100
0.49 117
0.36 123
0.36 136
7
Experiment 1 ATP'
"Ca Effluxt Experiment 2 ATP
'Ca Efflux
0.97 57
0.12 71
Experiment 3 ATP
%a Efflux ATP pmoles/ml RBC.
1.44 76
t Nanomoles YCa/min/lO1O ghosts.
0.75 71
.56 56
.49
88
.05 65
160
SCHRIER ET AL.
Table 2. UCa Associated with Ghost Membranes Length of Storage (weeks)
0
Experiment 1 Experiment 2 Experiment 3
25' 17 28
Nanomoles %a per
1.53
4
5
6
7
32 42 42 35 34 20 21 25 28 31 lO1O
ghosts.
37 C." Therefore residual 45Caadherent to ghost erythrocyte membranes was determined after 30 minutes of incubation at 37 C, and was not increased in ghosts prepared from stored erythrocytes (Table 2). Endocytosis in resealed red blood cell ghosts was decreased by five weeks of storage as measured by transmission electronmicroscopy (Fig. 2). This decrease in endocytosis could not be explained by inefficient resealing of ATP since following the resealing procedure the ghosts prepared from fresh erythrocytes contained 2.65 pmoles ATP per ml of packed ghosts while the
Tranifuiion March-April 1979
comparable ATP value for the samples stored five weeks was 3.26. However, when the erythrocytes from the five week storage sample were incubated with PIGPA and then studied for ghost endocytosis it was clear that endocytosis had returned to normal. Intact Erythrocytes Drug-induced endocytosis in intact erythrocytes was studied during the storage period both before and after PIGPA mediated restoration of ATP levels. In the single experiment shown in Figures 3 , 4 and 5 the extent of endocytosis was recorded as the increment of 57Co-VitaminB,, trapped in endocytic vacuoles per ml of RBC. Vinblastine used at two concentrations was not significantly affected by storage, and ATP restoration produced no measurable changes (Fig. 3). In this experiment chlorpromazine endocytosis became impaired by the third week of storage and deteriorated further but was never entirely inhibited. ATP restoration had no persistent effect on chlorpromazineendocytosisin this experiment (Fig. 4). Primaquine endocytosis was tested at
FIGS.2 A-C. Endocytosis induced by resealing 1 mM Ca and 2.5 mM Mg-ATP into ghosts. Fresh sample (A). A distinct reduction in red blood cell ghost endocytosis after storage for five weeks (B). Five week stored incubated for one hour with the PIGPA medium endocytosis (C) ~ 4 8 0 0 .
.
FIG.2B.
See legend for Figure 2A.
FIG.2C. See legend for Figure 2A.
162
Transfurion March-April 1979
SCHRIER ET AL.
minimally affected by storage, chlorpromazine 7 endocytosis in this experiment fell 16 per cent ATPRESrOREO
l-
I1
-0-
UNRESWD
A
to
of base line values after five weeks but could be restored fully after ATP rejuvenation. Primaquine endocytosis became undetectable at three weeks and could be restored to 60-65 per cent of base line values at five and four weeks of storage. SDS-PAGE analysis of ghosts before and after ATP restoration after six and seven weeks of storage revealed no consistent abnormalities on either gross inspection of the gels or on densitometric tracings (Fig. 6).The gels only are shown.
1
600
-
1
---
1
1
1
1
ATP RESTORED UNRESTORED
a 0
1
I
I
1
1
I
1
1
2
3
4
5
6
7
WEEKS STORAGE FIG.3. Vinblastine endocytosis in intact erythrocytes as the increment in cpm of "Co-Vitamin B,* trapped in one ml of erythrocytes. Two concentrations of Vinblastine were used. 0.5mM and 1.5mM. The dotted line indicates that those samples had their ATP levels restored prior to induction of endocytosis.
two concentrations and became essentially undetectable after the third week of storage (Fig. 5). Restoration of ATP brought primaquine endocytosis back to a maximum of 25 per cent of base line values. In six other experiments, (Table 3) primaquine endocytosis became reproducibly impaired by the third week of storage while ATP levels were still 25 to 75 per cent of base line values. In the experiment with Ju (Table 3), restoration of ATP levels at five weeks brought primaquine endocytosis from zero to 44 per cent of base line values. An additional experiment showing drug induced endocytosis before and after ATP restoration is shown in Table 4. Vinblastine endocytosis was
0
1
2
3
4
5
6
WEEKS STORAGE FIG.4. Endocytosis in intact erythrocytes induced with 0.9mM chlorpromazine. The symbols are as indicated in Fig. 3.
7
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MEMBRANE STORAGE LESION
increase during storage,6 we observed that there was no loss of activity of the 45Ca efflux pump and no increase in membrane associated calcium. The efflux data reported for red blood cell ghosts averages approximately 84 nanomoles per minute per 1O1O ghosts with arange of 56 to 136. These values are in reasonable agreement for Sarkadi's value for maximum "Ca efflux from intact red blood cells of approximately 85 nanomoles per minute per ml of packed RBC.'O Ghost endocytosis was impaired by five weeks of storage, and the impairment could not be explained by inefficient resealing of ATP into ghosts, or by abnormalities of the 45Capump or by increased 45Cabinding to ghost membranes. When ghosts were made from the same five week stored RBCs, in which the ATP levels had been restored for one hour, ghost endocytosis had returned to normal. This prompt reversibility of the impairment in ghost endocytosis upon restoration of erythrocytic ATP levels suggested that an ATP mediated membrane function was involved. Our measurements did not include studies of erythrocyte protein kinase or of the extent of membrane polypeptide phosphorylation. However, it is a reasonable hypothesis that ATP depletion might interfere with red blood cell ghost endocytosis by modifying the extent of erythrocyte membrane protein phosphorylation.2 Erythrocyte membrane polypeptides were characterized according to the reasonably
WEEKS SToRA# FIG.5. Endocytosis was induced by using 2mM and 3mM primaquine at the storage periods indicated. Symbols as in Fig. 3.
Table 3. Effects of Storage on Primequine Endocytosis Subjects
Ju
JA
GA
Weeks of Storage
ATP
Endocytosis
ATP
Endocytosis
3
.75
0'
.65
13'
4 5
56 .49
0 0/44%f
.44
.42
25 0
ATP
.78
.62 51
Primaquine Endocytosis-% Control Value.
t Restored Value.
JO
BA
HC
Endocytosis
ATP
Endocytosis
ATP
Endocytosis
81'
51
-
11'
.49
14'
.28
0
.20
76
28 23
-
-
-
ATP
Endocytosis
.39 .36
0' 0
-
-
164
Tnnsfuaion
SCHRIER ET AL.
FIG.6. SDS-polyacrylamide-gel electrophoretic patterns of ghosts prepared from stored erythrocytes. The tube gel on the left was prepared from the five week storage sample; the second from the left shows the electrophoretic pattern of the five week storage sample after ATP restoration; the third tube from the left shows the six week storage sample; and the tube on the right shows the six week stored sample after ATP restoration.
standard SDS-PAGE technique and showed no specific or reproducible changes. Endocytosis in stored intact erythrocytes showed distinct changes. Initially it had been proposed that all forms of drug induced
MUrh-Apd 1979
endocytosis proceeded via common mechanisms,' but differences have become apparent. Modest ATP depletion drastically inhibits primaquine endocytosis, while even severe ATP depletion does not totally block chlorpromazineor vinblastine induced endocytosi~.'~ The ionophore induced entry of calcium into erythrocytes stimulates primaquine endocytosis, inhibits vinblastine endocytosis, and does not affect chlorpromazine endocytosi~.~~ Storage did not affect vinblastine endocytosis, but it variably reduced but did not completely block chlorpromazine endocytosis and totally blocked primaquine endocytosis generally by three weeks at which time ATP levels were approximately half of normal. Restoration of ATP levels led to a variable improvement in chlorpromazine endocytosis but did not result in complete correction of primaquine endocytosis. Primaquine endocytosis is that form of drug endocytosis that most clearly requires preservation of substantial amounts of ATP.I3 However, in erythrocytes subjected to storage which produced only modest reduction of ATP, the impairment in primaquine endocytosis was persistent and subsequent restoration of ATP levels did not restore primaquine endocytosis to base line values. It appears that an erythrocytic membrane storage lesion occurs and that it can be studied quantitatively by the methods described. It is now necessary to determine if the membrane defect described leads to or correlates with impaired in vivo survival of erythrocytes.
Table 4. Drug-Induced Endocytosis before and after ATP Restoration ~
ATP Weeks Storage ATP' 8 after PIGPA
Prirnaquine 2 mM Unrestored
Initial Value
1.44
3 4 5
.75 .56 .49
ATP pmoles/ml RBC.
t After PIGPA.
2.25 1.44 1.57
Chlorprornazine 1.2 mM
Vinblastine 1.5 mM
Unrestored Unrestored 1ooOh Restoredt 1ooOh Restored?
100%
Restoredt
0 0 0
130
46
60
80
60
16
77 37 111
123 150 72
163 136 171
Volume 19
MEMBRANE STORAGE LESION
Number 2
References 1. Ben-Bassat, t., K. G. Bensch, and S. L. Schrier:
Drug-induced erythrocyte membrane internalization. J. Clin. Invest. 51:1833, 1972. 2. Birchmeier, W., and S. J. Singer: On the mechanism of ATP-induced shape changes in human erythrocyte membranes. J. Cell Biol. 73647, 1977. 3. Conrad, M. J., and J. T. Penniston: Surface proteins of the erythrocyte membrane: Effect of aging. Vox Sang. 26:1, 1974. 4. Dern, R. J., G. J. Brewer, and J. J. Wiorkowski: Studies on the preservation of human blood. 11. The relationship of erythrocyte adenosine triphosphate levels and other in vifro measures to red cell stoiagcability. J. Lab. Clin. Med. 69968, 1967. 5. Fairbanks, G.. T. L. Steck, and D. F. H. Wallach: Electrophoretic analysis of the major polypep tides of the human erythrocyte membrane. Biochemistry 10:2606, 1971. 6. Haradin, A. R., R. I. Weed, and C. F. Reed: Changes in physical properties of stored erythrocytes, relationship to survival in vivo. Transfusion 9229, 1969. 7. Hogman, C. F., 0. Akerblom, G. Arturson, C. H. de Verdier. A. Krueger, and M. Westman: Experiences with new preservatives: summary of experiments in Sweden. I n : The Human Red Cell in V i m . T. J. Greenwalt and G. A. Jamieson, Eds., New York, Gruneand Stratton, 1974,p. 217. 8. Meyerstein, N.. D. Mazor, and A. Dvilansky: Changes in agglomeration of human red blood cells in liquid storage in CPD media. Transfusion 17:llS. 1977. 9. Momson, M.:Discussion of paper. I n : The Human Red Cellin Virro. T. J. Greenwalt and G. A. Jamieson, Eds.. New York. Grune and Stratton, 1974,p. 83. 10. Sarkadi, B.. 1. Szasz, A. Gerloczy, and G. Gardos: Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells. Biochim. Biophys. Acta W 9 3 , 1977. 11. Schrier. S. L.,and K. G. Bensch: Endocytosis in resealed human erythrocyte ghosts: Abnormalities in sickle cell anemia. I n : Proceedings of the International Conference on Biological
165
Membranes. Membranes and Disease. L. Bolis, J. Hoffman, and A. Leaf, Eds.. New York. Rosen Press, 1976,p. 31. 12. -, K. G. Bensch, M. Johnson, and I. Junga: Energized endocytosis in human erythrocyte ghosts. J. Clin. Invest. 56:8, 1975. 13. -, I. Junga, J. Krueger, and M. Johnson: Requirements of drug induced endocytosis by intact human erythrocytes. Blood Cells 1978 4:339, 1978. I. Junga, and M.Seeger: The mechanism of 14. -, drug induced erythrocyte vacuole formation. J. Lab. Clin. Med. 83:215, 1974. 15. -, J. Krueger, I. Junga, and B. Hardy: RBC membrane storage lesion. Blood 5OSuppl. 310, 1977. 16. Valeri, C. R.: Metabolic regeneration of depleted erythrocytes and their frozen storage. I n : The Human Red Cell in Vitro. T. J. Greenwalt and G. A. Jamieson, Eds., New York, Grune and Stratton, 1974,p. 281. 17. Weed, R. J.. P. L. La Celle, and M. Udkaw: Structure and function of the red cell membrane: Changes during storage. I n : The Human Red Cell in Virro. T. J. Greenwalt and G. A. Jamieson, Eds. New York, Grune and Stratton, 1974,p. 65. 18. Zuck, T. F., T. A. Bensinger, C. C. Peck, R. K. Chillar, E. Beutler, L. N. Button, P. R. McCurdy, A. M.Josephson, and T. J. Greenwak The in vivo survival of red blood cells stored in modified CPD with adenine: Report of a multiinstitutional cooperative effort. Transfusion 11:374, 1977.
Stanley L. Schrier, M.D., Professor of Medicine (Hematology),Chief, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305. Britta Hardy, Ph.D., Fellow in Hematology, Division of Hematology. Klaus Bensch, M.D.,Professor of Pathology, Department of Pathology. Irene G. Junga, B.S., Senior Research Assistant, Division of Hematology. Judy A. Krueger, B.S., Research Assistant, Division of Hematology.
Red Blood Cell Membrane Storage Lesion S. L. SCHRIER,B. HARDY,K. BENSCH,I. JUNGAA N D J. KRUEGER From the Departments of Hematology and Pathology. Stanford University School of Medicine, Stanford, California StORep d human erythmcytesin CPD produced a lesion of the erythrocyte membrane manifested by abnormal endocytosb in resealed ghosts and in intact erythrocytes. Endocytosis produced by rescaling Ca, Mg, and ATP into g b t a was impaired by Rve weeks of storage and this defect was promptly reversed by the prior repneratbn of ATP in the stored erytbmcytes. DrugInduced endocytods was studied in intact stored erythrocytes. VinblPstine endocytosis was not affected by storage. Chlorpromazlne endocytmls was variably but never compktely inhibited by storage, and restoration of ATP occasbndy resulted in compkte restoration of chlorpromsdne endocytosis to base line values. However, prlmaquine endocytosis was usually totally inhibited after three to four weeks of storage at a time when residual ATP kvels were 30 to 50 per cent of baae Une values. Restoration of ATP levels to at kast baae line values dM not compktely restore primaquine endocytosls to control values. Study of primaquine endocytosrs provides an opportunity for delning an erythrocyte membrane storage kslon.
A VARIETY of methods have been used to extend the allowable blood bank storage period for erythrocytes. Supplementation of the anticoagulant solution has been extensively studied with the aim of avoiding the metabolic storage lesion which usually means preserving relatively high levels of ATP, and ~ , ~ - D P G . 'Persistence B'~ of ATP correlates imperfectly with the 24 hour posttransfusion survival of erythrocyte^,^.'^ however, the mechanisms by which ATP might work have not been clearly established. Since ATP has known membrane functions, it is possible that ATP depletion induced by storage, could affect erythrocyte membrane functions." Membrane storage lesions have been described. These have Supported by NIH Grant AM 13682. A preliminary report of this work was presented to the American Society of Hematology. Received for publication January 23, 1978; accepted February 27, 1978.
included alterations in membrane polypept i d e ~enhanced ,~ accessability of membrane proteins to external probes,Band increased agglomeration of erythrocytes in media of low ionic strength at pH 7.2.8 Theoretically, if a membrane storage lesion developed, perhaps as a consequence of prolonged ATP depletion, it could ultimately limit the shelf life of erythrocytes stored in liquid media. We therefore proposed to determine if there was a functional erythrocyte membrane storage lesion by measuring endocytosis in vitro in intact erythrocytes' and in their resealed ghosts1* because endocytosis involves a variable interaction between ATP and the membrane functions of invagination and fusion.'J9 Materials and Methods Seven normal donors participated in various aspects of the experiments according to protocols and informed consent forms approved by the Stanford University Committee on Human Experimentation. Venous blood was drawn directly into CPD contained in sterile 16 mm x 125 mm Falcon Plastic Screw-top Tubes using a ratio of 0.14 ml of CPD per ml of whole blood. The anticoagulated blood was stored at 4 C and the tubes were inverted once at weekly intervals. ATP levels in erythrocytes were determined by the coupled hexokinase-glucose-6-phosphate dehydrogenase reaction in neutralized perchloric acid filtrates and reported as p moles per ml of packed RBC. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on "white Dodge ghosts'' by the method of Fairbanks. Endocytosis in intact erythrocytes was induced by incubation with primaquine. chlorpromazine, and vinblastine using the materials and methods previously described.**14 In intact erythrocytes, endocytosis was evaluated by
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MEMBRANE STORAGE LESION
phase microscopy and by trapping of T o Vitamin BI2within endocytic vacuole^.^.^^ Endocytosis in red blood cell ghosts was induced by resealing 2.5 mM Mg-ATP and 1.0 mM 'Va within the ghosts.'* Measurement of red blood cell ghost endocytosis was achieved by phase and transmission electron microscopy (TEM). We also measured ' T a efflux. and the amount of residual 45Ca binding to red ghost membranes." Because there is a degree of variability in measuring quantitative aspects of endocytosis the experiments were usually designed so that blood was withdrawn from the normal donor at stipulated times before the final day on which all the experiments were performed. Therefore, the endocytosis data for a single experiment is derived from measurements made on one day using the same reagents and controls. Restoration of ATP levels was achieved by incubating erythrocytes at 37 C for one hour with the pyruvate-inosine-glucose-phosphate-adenine (PIGPA) medium exactly as described by Valeri.Is
ATP CONTENT OF R E
---
Results .I
ATP Content of Erythrocytes The data for one experiment are shown in Figure 1. Incubation with the PIGPA medium resulted in the restoration of ATP levels at least to base line values.
0
I
EsTmEOBT )IIQIIlTnN 2
s
4
c
=pCo#.
S
6
WEEKS SrORlyT
FIG.1. ATPlevels plotted against storage in weeks (solid line). The dotted line indicates the ATP values after the samples had been incubated for one hour with the PIGPA medium.
Resealed Red Blood Cell Ghosts We had previously reported that in red blood cell ghosts there was a correlation between endocytosis, and activity of Ca, Mg-ATPase and *Ca efflux.'* Therefore, 'Ta efflux was measured in red blood cell ghosts made from stored erythrocytes and no impairment in 45Caeftlux
was observed (Table 1). In sickle cell anemia there is both deficient ghost endocytosis and a five-fold increase above normal in the amount of 45Ca remaining adherent to erythrocyte ghost membranes after 30 minutes of incubation at
Table 1. %a €ff/ux from Resealed Ghosts Length of Storage (weeks)
0
1.5
3
4
5
6
1.39 100
0.89 117
0.68 100
0.49 117
0.36 123
0.36 136
7
Experiment 1 ATP'
"Ca Effluxt Experiment 2 ATP
'Ca Efflux
0.97 57
0.12 71
Experiment 3 ATP
%a Efflux ATP pmoles/ml RBC.
1.44 76
t Nanomoles YCa/min/lO1O ghosts.
0.75 71
.56 56
.49
88
.05 65
160
SCHRIER ET AL.
Table 2. UCa Associated with Ghost Membranes Length of Storage (weeks)
0
Experiment 1 Experiment 2 Experiment 3
25' 17 28
Nanomoles %a per
1.53
4
5
6
7
32 42 42 35 34 20 21 25 28 31 lO1O
ghosts.
37 C." Therefore residual 45Caadherent to ghost erythrocyte membranes was determined after 30 minutes of incubation at 37 C, and was not increased in ghosts prepared from stored erythrocytes (Table 2). Endocytosis in resealed red blood cell ghosts was decreased by five weeks of storage as measured by transmission electronmicroscopy (Fig. 2). This decrease in endocytosis could not be explained by inefficient resealing of ATP since following the resealing procedure the ghosts prepared from fresh erythrocytes contained 2.65 pmoles ATP per ml of packed ghosts while the
Tranifuiion March-April 1979
comparable ATP value for the samples stored five weeks was 3.26. However, when the erythrocytes from the five week storage sample were incubated with PIGPA and then studied for ghost endocytosis it was clear that endocytosis had returned to normal. Intact Erythrocytes Drug-induced endocytosis in intact erythrocytes was studied during the storage period both before and after PIGPA mediated restoration of ATP levels. In the single experiment shown in Figures 3 , 4 and 5 the extent of endocytosis was recorded as the increment of 57Co-VitaminB,, trapped in endocytic vacuoles per ml of RBC. Vinblastine used at two concentrations was not significantly affected by storage, and ATP restoration produced no measurable changes (Fig. 3). In this experiment chlorpromazine endocytosis became impaired by the third week of storage and deteriorated further but was never entirely inhibited. ATP restoration had no persistent effect on chlorpromazineendocytosisin this experiment (Fig. 4). Primaquine endocytosis was tested at
FIGS.2 A-C. Endocytosis induced by resealing 1 mM Ca and 2.5 mM Mg-ATP into ghosts. Fresh sample (A). A distinct reduction in red blood cell ghost endocytosis after storage for five weeks (B). Five week stored incubated for one hour with the PIGPA medium endocytosis (C) ~ 4 8 0 0 .
.
FIG.2B.
See legend for Figure 2A.
FIG.2C. See legend for Figure 2A.
162
Transfurion March-April 1979
SCHRIER ET AL.
minimally affected by storage, chlorpromazine 7 endocytosis in this experiment fell 16 per cent ATPRESrOREO
l-
I1
-0-
UNRESWD
A
to
of base line values after five weeks but could be restored fully after ATP rejuvenation. Primaquine endocytosis became undetectable at three weeks and could be restored to 60-65 per cent of base line values at five and four weeks of storage. SDS-PAGE analysis of ghosts before and after ATP restoration after six and seven weeks of storage revealed no consistent abnormalities on either gross inspection of the gels or on densitometric tracings (Fig. 6).The gels only are shown.
1
600
-
1
---
1
1
1
1
ATP RESTORED UNRESTORED
a 0
1
I
I
1
1
I
1
1
2
3
4
5
6
7
WEEKS STORAGE FIG.3. Vinblastine endocytosis in intact erythrocytes as the increment in cpm of "Co-Vitamin B,* trapped in one ml of erythrocytes. Two concentrations of Vinblastine were used. 0.5mM and 1.5mM. The dotted line indicates that those samples had their ATP levels restored prior to induction of endocytosis.
two concentrations and became essentially undetectable after the third week of storage (Fig. 5). Restoration of ATP brought primaquine endocytosis back to a maximum of 25 per cent of base line values. In six other experiments, (Table 3) primaquine endocytosis became reproducibly impaired by the third week of storage while ATP levels were still 25 to 75 per cent of base line values. In the experiment with Ju (Table 3), restoration of ATP levels at five weeks brought primaquine endocytosis from zero to 44 per cent of base line values. An additional experiment showing drug induced endocytosis before and after ATP restoration is shown in Table 4. Vinblastine endocytosis was
0
1
2
3
4
5
6
WEEKS STORAGE FIG.4. Endocytosis in intact erythrocytes induced with 0.9mM chlorpromazine. The symbols are as indicated in Fig. 3.
7
Volume 19 Number 2
163
MEMBRANE STORAGE LESION
increase during storage,6 we observed that there was no loss of activity of the 45Ca efflux pump and no increase in membrane associated calcium. The efflux data reported for red blood cell ghosts averages approximately 84 nanomoles per minute per 1O1O ghosts with arange of 56 to 136. These values are in reasonable agreement for Sarkadi's value for maximum "Ca efflux from intact red blood cells of approximately 85 nanomoles per minute per ml of packed RBC.'O Ghost endocytosis was impaired by five weeks of storage, and the impairment could not be explained by inefficient resealing of ATP into ghosts, or by abnormalities of the 45Capump or by increased 45Cabinding to ghost membranes. When ghosts were made from the same five week stored RBCs, in which the ATP levels had been restored for one hour, ghost endocytosis had returned to normal. This prompt reversibility of the impairment in ghost endocytosis upon restoration of erythrocytic ATP levels suggested that an ATP mediated membrane function was involved. Our measurements did not include studies of erythrocyte protein kinase or of the extent of membrane polypeptide phosphorylation. However, it is a reasonable hypothesis that ATP depletion might interfere with red blood cell ghost endocytosis by modifying the extent of erythrocyte membrane protein phosphorylation.2 Erythrocyte membrane polypeptides were characterized according to the reasonably
WEEKS SToRA# FIG.5. Endocytosis was induced by using 2mM and 3mM primaquine at the storage periods indicated. Symbols as in Fig. 3.
Table 3. Effects of Storage on Primequine Endocytosis Subjects
Ju
JA
GA
Weeks of Storage
ATP
Endocytosis
ATP
Endocytosis
3
.75
0'
.65
13'
4 5
56 .49
0 0/44%f
.44
.42
25 0
ATP
.78
.62 51
Primaquine Endocytosis-% Control Value.
t Restored Value.
JO
BA
HC
Endocytosis
ATP
Endocytosis
ATP
Endocytosis
81'
51
-
11'
.49
14'
.28
0
.20
76
28 23
-
-
-
ATP
Endocytosis
.39 .36
0' 0
-
-
164
Tnnsfuaion
SCHRIER ET AL.
FIG.6. SDS-polyacrylamide-gel electrophoretic patterns of ghosts prepared from stored erythrocytes. The tube gel on the left was prepared from the five week storage sample; the second from the left shows the electrophoretic pattern of the five week storage sample after ATP restoration; the third tube from the left shows the six week storage sample; and the tube on the right shows the six week stored sample after ATP restoration.
standard SDS-PAGE technique and showed no specific or reproducible changes. Endocytosis in stored intact erythrocytes showed distinct changes. Initially it had been proposed that all forms of drug induced
MUrh-Apd 1979
endocytosis proceeded via common mechanisms,' but differences have become apparent. Modest ATP depletion drastically inhibits primaquine endocytosis, while even severe ATP depletion does not totally block chlorpromazineor vinblastine induced endocytosi~.'~ The ionophore induced entry of calcium into erythrocytes stimulates primaquine endocytosis, inhibits vinblastine endocytosis, and does not affect chlorpromazine endocytosi~.~~ Storage did not affect vinblastine endocytosis, but it variably reduced but did not completely block chlorpromazine endocytosis and totally blocked primaquine endocytosis generally by three weeks at which time ATP levels were approximately half of normal. Restoration of ATP levels led to a variable improvement in chlorpromazine endocytosis but did not result in complete correction of primaquine endocytosis. Primaquine endocytosis is that form of drug endocytosis that most clearly requires preservation of substantial amounts of ATP.I3 However, in erythrocytes subjected to storage which produced only modest reduction of ATP, the impairment in primaquine endocytosis was persistent and subsequent restoration of ATP levels did not restore primaquine endocytosis to base line values. It appears that an erythrocytic membrane storage lesion occurs and that it can be studied quantitatively by the methods described. It is now necessary to determine if the membrane defect described leads to or correlates with impaired in vivo survival of erythrocytes.
Table 4. Drug-Induced Endocytosis before and after ATP Restoration ~
ATP Weeks Storage ATP' 8 after PIGPA
Prirnaquine 2 mM Unrestored
Initial Value
1.44
3 4 5
.75 .56 .49
ATP pmoles/ml RBC.
t After PIGPA.
2.25 1.44 1.57
Chlorprornazine 1.2 mM
Vinblastine 1.5 mM
Unrestored Unrestored 1ooOh Restoredt 1ooOh Restored?
100%
Restoredt
0 0 0
130
46
60
80
60
16
77 37 111
123 150 72
163 136 171
Volume 19
MEMBRANE STORAGE LESION
Number 2
References 1. Ben-Bassat, t., K. G. Bensch, and S. L. Schrier:
Drug-induced erythrocyte membrane internalization. J. Clin. Invest. 51:1833, 1972. 2. Birchmeier, W., and S. J. Singer: On the mechanism of ATP-induced shape changes in human erythrocyte membranes. J. Cell Biol. 73647, 1977. 3. Conrad, M. J., and J. T. Penniston: Surface proteins of the erythrocyte membrane: Effect of aging. Vox Sang. 26:1, 1974. 4. Dern, R. J., G. J. Brewer, and J. J. Wiorkowski: Studies on the preservation of human blood. 11. The relationship of erythrocyte adenosine triphosphate levels and other in vifro measures to red cell stoiagcability. J. Lab. Clin. Med. 69968, 1967. 5. Fairbanks, G.. T. L. Steck, and D. F. H. Wallach: Electrophoretic analysis of the major polypep tides of the human erythrocyte membrane. Biochemistry 10:2606, 1971. 6. Haradin, A. R., R. I. Weed, and C. F. Reed: Changes in physical properties of stored erythrocytes, relationship to survival in vivo. Transfusion 9229, 1969. 7. Hogman, C. F., 0. Akerblom, G. Arturson, C. H. de Verdier. A. Krueger, and M. Westman: Experiences with new preservatives: summary of experiments in Sweden. I n : The Human Red Cell in V i m . T. J. Greenwalt and G. A. Jamieson, Eds., New York, Gruneand Stratton, 1974,p. 217. 8. Meyerstein, N.. D. Mazor, and A. Dvilansky: Changes in agglomeration of human red blood cells in liquid storage in CPD media. Transfusion 17:llS. 1977. 9. Momson, M.:Discussion of paper. I n : The Human Red Cellin Virro. T. J. Greenwalt and G. A. Jamieson, Eds.. New York. Grune and Stratton, 1974,p. 83. 10. Sarkadi, B.. 1. Szasz, A. Gerloczy, and G. Gardos: Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells. Biochim. Biophys. Acta W 9 3 , 1977. 11. Schrier. S. L.,and K. G. Bensch: Endocytosis in resealed human erythrocyte ghosts: Abnormalities in sickle cell anemia. I n : Proceedings of the International Conference on Biological
165
Membranes. Membranes and Disease. L. Bolis, J. Hoffman, and A. Leaf, Eds.. New York. Rosen Press, 1976,p. 31. 12. -, K. G. Bensch, M. Johnson, and I. Junga: Energized endocytosis in human erythrocyte ghosts. J. Clin. Invest. 56:8, 1975. 13. -, I. Junga, J. Krueger, and M. Johnson: Requirements of drug induced endocytosis by intact human erythrocytes. Blood Cells 1978 4:339, 1978. I. Junga, and M.Seeger: The mechanism of 14. -, drug induced erythrocyte vacuole formation. J. Lab. Clin. Med. 83:215, 1974. 15. -, J. Krueger, I. Junga, and B. Hardy: RBC membrane storage lesion. Blood 5OSuppl. 310, 1977. 16. Valeri, C. R.: Metabolic regeneration of depleted erythrocytes and their frozen storage. I n : The Human Red Cell in Vitro. T. J. Greenwalt and G. A. Jamieson, Eds., New York, Grune and Stratton, 1974,p. 281. 17. Weed, R. J.. P. L. La Celle, and M. Udkaw: Structure and function of the red cell membrane: Changes during storage. I n : The Human Red Cell in Virro. T. J. Greenwalt and G. A. Jamieson, Eds. New York, Grune and Stratton, 1974,p. 65. 18. Zuck, T. F., T. A. Bensinger, C. C. Peck, R. K. Chillar, E. Beutler, L. N. Button, P. R. McCurdy, A. M.Josephson, and T. J. Greenwak The in vivo survival of red blood cells stored in modified CPD with adenine: Report of a multiinstitutional cooperative effort. Transfusion 11:374, 1977.
Stanley L. Schrier, M.D., Professor of Medicine (Hematology),Chief, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305. Britta Hardy, Ph.D., Fellow in Hematology, Division of Hematology. Klaus Bensch, M.D.,Professor of Pathology, Department of Pathology. Irene G. Junga, B.S., Senior Research Assistant, Division of Hematology. Judy A. Krueger, B.S., Research Assistant, Division of Hematology.
