Platelet Glycolysis in Platelet Storage 111. The Inability of Platelets to Utilize Exogenous Citrate C. TEGOSA N D E. BEUTLER From the Department of Hematology, City of Hope Medical Center, Duarte. California
The metabolism of exogenow citrate by freshly c o k t e d human platelets has been investigated by measuring the disappearance of citrate from platelet suspensions and by measuring the formation of COz and glycogen from purified radioactive citrate. These studies indicate that platelets do not have the capacity to metabolizeexogenous citrate.
THEINCREASING IMPORTANCE of platelet preservation has directed our attention to the energy metabolism of platelets. Since platelets are generally stored at room temperature, they rapidly consume the primary source of cellular energy, glucose. If the citrate used to prevent coagulation of blood could be metabolized by the platelets, it could serve as an important energy source during storage. Karpatkin3 reported that in the presence of citrate, platelets manifested higher ATP levels and suggested that the citrate was metabolized. Moreover, in a subsequent study, the same authop claimed that 14Clabeled citrate was incorporated into platelet glycogen. We now show, however, that platelets are unable to consume exogenous citrate or to metabolize it to either CO, or glycogen. Materials and Methods Blood was collected in standard citrate-phosphate-dextrose (CPD) solution' and processed at room temperature. Platelet-rich plasma was prepared by centrifugation at 1,000 x g for nine minutes as described by Slichter and Harker.6 The platelets were then sedimented by centrifugaReceived for publication August 17, 1978; accepted October 2, 1978. Supported in part by grant HE/07449 from the National Institutes of Health.
tion at 3,000 x g for ten minutes. The plateletpoor plasma was removed and the platelets were permitted to disaggregate at room temperature for one to two hours. They were then resuspended in 40 ml of platelet-poor plasma and freed of white blood cells by centrifugation at 800 x g for nine minutes. The platelets and the platelet concentrates were then washed two times in a washing buffer containing 30 mM triethanolamine-HCI, pH 7.8 containing 123 mM NaCl, 0.2 mM KH2P04, 0.8 mM K2HP04 and 5 mM ethylenediaminetetraaceticacid (EDTA). Unless otherwise indicated, the platelets were then suspended in the washing buffer at a protein concentration of between 0.7 and 0.8 mglml. 14C-citricacid was purchased from Amersham Corp. IL. Purified 14C-citricacid was prepared by applying the commercial citric acid to a 1.5 x 25 cm Dowex-1 chloride column, washing it with water, and eluting with a linear 1,OOO ml 0 to 0.2 M HCI gradient. About 0.5 per cent of the radioactivity ran through the column. Another 2.0 per cent of the radioactivity eluted just before the main peak of citric acid, in a position which was shown by enzymatic analysis to contain isocitric acid, and almost all of the remaining radioactivity was eluted as a symetrical major peak which was pooled and lyophilized.The lyophilized material chromatographed as a single peak. Citrate analyses were carried out on boiled extracts by a modification of the system described by Sieberg.5Citric acid was converted to isocitric acid with aconitase and the conversion of NADP+ to NADPH was measured at 340 nm in the presence of isocitric dehydrogenase and aconitase. The complete system contained in a 1 ml system: Tris-HC1 5 mM EDTA buffer, pH 8.0, 100 pl; MgS04, 20 mM, 100 pl; NaK tartrate, 0.2 M, 100 pl; NADP, 2 mM, 100 pl; isocitric dehydrogenase (Sigma) 44 U/ml 5 p1; boiled extract and H20 to make 0.99 ml. The reaction was started by the addition of 10 p1 "activated" aconitase. Activation of aconitase was achieved by adding 10 mg of aconitase (Sigma) in 200 pl Tris-HCI, 0.5 M, pH 8.0 to 10 p1 1 mM ferrous ammonium sulfate and 50 mM 20 pl cysteine-HC1.
0041-1 132/79/0900/0601 $00.65 0 J. B. Lippincott C o . Transfusion September-October 1979
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Table 1. Citrate Levels in Platelet Suspensions Incubated with and without Citrate
Added Citrate
0 rnM
0.45 rnM
Transfusion
TEGOS AND BEUTLER
Time (rnin.)
Citrate Concentrations (Mean ? SE; n = 3)
0 60 120
0.082 t 0.002 rnM 0.111 2 0.006 rnM 0.130 ? 0.005 rnM
0 60 120
0.564 2 0.027 rnM 0.606 c 0.002 rnM 0.646 c 0.026 rnM
To measure CO, evolution 2 ml portions of the washed platelet suspension were resuspended in the washing buffer containing 0.5 mM citrate and 0.2 pCi “C-citrate and were placed in the main chamber of the Warburg flask. The center well contained Hyamine (p-(diisobutylcresoxyethoxyethyl) dimethylbenzylammonium hydroxide) and the sidearm one ml of 10% perchloric acid. The reaction was stopped immediately or after 60 or 120 minutes of incubation at 37 C in a metabolic shaker by tipping the perchloric acid into the platelet suspension. Shaking was continued for two hours and radioactivity in the Hyamine counted. To measure incorporation of radioactive citrate into glycogen, the incubation system described by Karpatkin was used.‘ Approximately 6 x 10” platelets were suspended in a 3.5 ml of Ringers bicarbonate buffer containing 4 mM KCI and 117 mM NaCI, 20 mM HaHCO,, 2 mM Na,S04, 2 mM N+HFQ, and 2 mM EDTA with the pH adjusted to 7.1. After 0.2 FC of I4C citrate with a specific activity of 20 pCilmMol were added to the buffer incubation was carried out at 37 C. Glycogen was isolated from platelets by alkaline extraction and ethanol precipitation., Results Consumption of Citric Acid by Platelets Platelets suspended in washing buffer were incubated with and without addition of 450 p M
citrate. The citric acid levels of these platelet suspensions were determined at 0, 60 and 120 minutes’ incubation. The results of these studies are summarized in Table 1. No consumption of citric acid occurred. Indeed, there was net appearance of citric acid both in the absence and the presence of exogenous citric acid. Evolution of CO, from Labeled Citric Acid When platelets were incubated with com-
mercially available citrate, variable amounts of
September-October I979
radioactivity were trapped in Hyamine. However, when chromatographically purified radioactive citrate was used as a substrate, only negligible quantities of radioactivity appeared in the center well of the incubating flask and there was no progressive increase of radioactivity with time. The results of these experiments are shown in Table 2. Appearance of Radioactivity in Glycogen
The radioactivity of glycogen isolated from platelets incubated with commercially available and purified citrate is shown in Table 3. Although a minute amount of radioactivity appeared in the “glycogen” fraction of platelets, most of this radioactivity proved to be dialyzable and therefore not actually glycogen.
Discussion
We have investigated the possible metabolism of exogenous citrate by platelets in three different systems by measuring: 1) the “disappearance” of citrate from suspensions of platelets incubated with citrate; 2) the evolution of CO, from platelets incubated with radioactive citrate; and 3) the incorporation of radioactivity from citric acid into platelet glycogen. None of these studies gives any indication that exogenous citric acid is metabolized by platelets. No citric acid was consumed by platelets. Indeed, the amount of citric acid increased progressively during incubation. Presumably, citric acid was synthesized from oxaloacetate and acetyl CoA formed from glycogen. Moreover, no evidence was obtained that appreciable amounts of citrate carbon found their way either into C o n ,the usual product of citrate metabolism by cells, or into glycogen as might occur in gluconeogenesis. Table 2. The 14C02 Production from Platelets during Incubation in the Presence of I4C-Citrate Incubation Time (rnin.)
Purified “C-Citrate
Nonpurified W-Citrate
0 60 120
0.053 ? 0.010’ 0.060 2 0.004 0.027 t 0.009
0.134 ? 0.087’ 0.116 c 0.040 0.201 c 0.060
Mean t SE in 4 experiments expressed as per cent of total radioactivity.
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Table 3. The Incorporation of “C-Citrate into Platelet Glycogen Purified “C-Citrate’ Incubation Time (min.) -~
0 60 120
Nondialysed Glycogen ~~
0.135 ? 0.013 0.151 2 0.023 0.155 f 0.001
Nonpurified IF-Citrate’
Dialysed Glycogen
Nondialysed Glycogen
Dialysed Glycogen
0.024 f 0.007 0.013 f 0.004 0.005 f 0.003
0.156 f 0.016 0.139 2 0.016 0.205 f 0.007
0.009 f 0.004 0.017 2 0.001 0.017 2 0.002
~
Results expressed as per cent of total radioactivity incorporated.
Our conclusions clearly differ from those previously presented by Karpatki~~.~.‘ His finding that citrate affected ATP levels of incubating platelets3 cannot be considered as evidence of citrate metabolism. Citrate has the capacity to influence metabolism in a variety of ways. It can chelate calcium, magnesium and other divalent cations. Moreover, because it is a strongly negatively charged polyanion at physiologic pH levels, it can influence intracellular pH through the Donnan membrane equilibrium effect. Karpatkin‘ found that tommercially available “C-citrate was incorporated into glycogen. Commercial preparations of radioisotopes are invariably contaminated with small amounts of other radioactive compounds. We found that citrate which was commercially available in 1978 to be no exception to this rule. Presumably, the commercially available citrate which Karpatkin employed was also contaminated with various radioactive compounds. Since the incorporation of I4C “citrate” into glycogen in Karpatkin’s studies was very modest, his findings are easily consistent with the incorporation of a contaminant. No significant incorporation of citrate into platelet glycogen was found in our investigations. We find that citrate is not metabolized by platelets. It is apparent that other sources
of metabolic energy must be made available during platelet storage. References 1. Anticoagulant citrate phosphate dextrose solution. The Pharmacopeia of the United States of America. The United States Pharmacopeial Convention, Inc. 48. Mack Publishing Company, Easton, PA, 1970. 2. Hassid, W. Z., and S. Abraham: Chemical procedures for analysis of polysaccharides. Method. Enzymol. 3:34, 1957. 3. Karpatkin, S.: Studies on human platelet glycolysis. Effect of glucose, cyanide, insulin, citrate, and agglutination and contraction on platelet glycolysis. J. Clin. Invest. 46:409, 1%7. 4. A. Charmatz, and R. M. Langer: Glycogenesis and glyconeogenesis in human platelets. Incorporation of glucose, pyruvate, and citrate into platelet glycogen: Glycogen synthetase and fructose-1,bdiphosphatase activity. J. Clin. Invest. 49: 140, 1970. 5 . Siebert, G.: Citrate and isocitrate. Determination with aconitase and isocitric dehydrogenase. I n : Methods of Enzymatic Analysis. H. U. Bergmeyer, Ed., New York, Academic Press. 1963, p. 318. 6. Slichter, S. J., and L. A. Harker: Preparation and storage of platelet concentrates. 1. Factors influencing the harvest of viable platelets from whole blood. Br. J. Haematol. M395, 1976.
-.
Constantinos Tegos, M.D., Junior Research Scientist, Hematology Department, City of Hope National Medical Center, Duarte, California 91010. Ernest Beutler, M.D., Chairman, Department of Clinical Research, Scripps Clinic & Research Foundation, La Jolla, California 92037.
The metabolism of exogenow citrate by freshly c o k t e d human platelets has been investigated by measuring the disappearance of citrate from platelet suspensions and by measuring the formation of COz and glycogen from purified radioactive citrate. These studies indicate that platelets do not have the capacity to metabolizeexogenous citrate.
THEINCREASING IMPORTANCE of platelet preservation has directed our attention to the energy metabolism of platelets. Since platelets are generally stored at room temperature, they rapidly consume the primary source of cellular energy, glucose. If the citrate used to prevent coagulation of blood could be metabolized by the platelets, it could serve as an important energy source during storage. Karpatkin3 reported that in the presence of citrate, platelets manifested higher ATP levels and suggested that the citrate was metabolized. Moreover, in a subsequent study, the same authop claimed that 14Clabeled citrate was incorporated into platelet glycogen. We now show, however, that platelets are unable to consume exogenous citrate or to metabolize it to either CO, or glycogen. Materials and Methods Blood was collected in standard citrate-phosphate-dextrose (CPD) solution' and processed at room temperature. Platelet-rich plasma was prepared by centrifugation at 1,000 x g for nine minutes as described by Slichter and Harker.6 The platelets were then sedimented by centrifugaReceived for publication August 17, 1978; accepted October 2, 1978. Supported in part by grant HE/07449 from the National Institutes of Health.
tion at 3,000 x g for ten minutes. The plateletpoor plasma was removed and the platelets were permitted to disaggregate at room temperature for one to two hours. They were then resuspended in 40 ml of platelet-poor plasma and freed of white blood cells by centrifugation at 800 x g for nine minutes. The platelets and the platelet concentrates were then washed two times in a washing buffer containing 30 mM triethanolamine-HCI, pH 7.8 containing 123 mM NaCl, 0.2 mM KH2P04, 0.8 mM K2HP04 and 5 mM ethylenediaminetetraaceticacid (EDTA). Unless otherwise indicated, the platelets were then suspended in the washing buffer at a protein concentration of between 0.7 and 0.8 mglml. 14C-citricacid was purchased from Amersham Corp. IL. Purified 14C-citricacid was prepared by applying the commercial citric acid to a 1.5 x 25 cm Dowex-1 chloride column, washing it with water, and eluting with a linear 1,OOO ml 0 to 0.2 M HCI gradient. About 0.5 per cent of the radioactivity ran through the column. Another 2.0 per cent of the radioactivity eluted just before the main peak of citric acid, in a position which was shown by enzymatic analysis to contain isocitric acid, and almost all of the remaining radioactivity was eluted as a symetrical major peak which was pooled and lyophilized.The lyophilized material chromatographed as a single peak. Citrate analyses were carried out on boiled extracts by a modification of the system described by Sieberg.5Citric acid was converted to isocitric acid with aconitase and the conversion of NADP+ to NADPH was measured at 340 nm in the presence of isocitric dehydrogenase and aconitase. The complete system contained in a 1 ml system: Tris-HC1 5 mM EDTA buffer, pH 8.0, 100 pl; MgS04, 20 mM, 100 pl; NaK tartrate, 0.2 M, 100 pl; NADP, 2 mM, 100 pl; isocitric dehydrogenase (Sigma) 44 U/ml 5 p1; boiled extract and H20 to make 0.99 ml. The reaction was started by the addition of 10 p1 "activated" aconitase. Activation of aconitase was achieved by adding 10 mg of aconitase (Sigma) in 200 pl Tris-HCI, 0.5 M, pH 8.0 to 10 p1 1 mM ferrous ammonium sulfate and 50 mM 20 pl cysteine-HC1.
0041-1 132/79/0900/0601 $00.65 0 J. B. Lippincott C o . Transfusion September-October 1979
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Volume 19 Number 5
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Table 1. Citrate Levels in Platelet Suspensions Incubated with and without Citrate
Added Citrate
0 rnM
0.45 rnM
Transfusion
TEGOS AND BEUTLER
Time (rnin.)
Citrate Concentrations (Mean ? SE; n = 3)
0 60 120
0.082 t 0.002 rnM 0.111 2 0.006 rnM 0.130 ? 0.005 rnM
0 60 120
0.564 2 0.027 rnM 0.606 c 0.002 rnM 0.646 c 0.026 rnM
To measure CO, evolution 2 ml portions of the washed platelet suspension were resuspended in the washing buffer containing 0.5 mM citrate and 0.2 pCi “C-citrate and were placed in the main chamber of the Warburg flask. The center well contained Hyamine (p-(diisobutylcresoxyethoxyethyl) dimethylbenzylammonium hydroxide) and the sidearm one ml of 10% perchloric acid. The reaction was stopped immediately or after 60 or 120 minutes of incubation at 37 C in a metabolic shaker by tipping the perchloric acid into the platelet suspension. Shaking was continued for two hours and radioactivity in the Hyamine counted. To measure incorporation of radioactive citrate into glycogen, the incubation system described by Karpatkin was used.‘ Approximately 6 x 10” platelets were suspended in a 3.5 ml of Ringers bicarbonate buffer containing 4 mM KCI and 117 mM NaCI, 20 mM HaHCO,, 2 mM Na,S04, 2 mM N+HFQ, and 2 mM EDTA with the pH adjusted to 7.1. After 0.2 FC of I4C citrate with a specific activity of 20 pCilmMol were added to the buffer incubation was carried out at 37 C. Glycogen was isolated from platelets by alkaline extraction and ethanol precipitation., Results Consumption of Citric Acid by Platelets Platelets suspended in washing buffer were incubated with and without addition of 450 p M
citrate. The citric acid levels of these platelet suspensions were determined at 0, 60 and 120 minutes’ incubation. The results of these studies are summarized in Table 1. No consumption of citric acid occurred. Indeed, there was net appearance of citric acid both in the absence and the presence of exogenous citric acid. Evolution of CO, from Labeled Citric Acid When platelets were incubated with com-
mercially available citrate, variable amounts of
September-October I979
radioactivity were trapped in Hyamine. However, when chromatographically purified radioactive citrate was used as a substrate, only negligible quantities of radioactivity appeared in the center well of the incubating flask and there was no progressive increase of radioactivity with time. The results of these experiments are shown in Table 2. Appearance of Radioactivity in Glycogen
The radioactivity of glycogen isolated from platelets incubated with commercially available and purified citrate is shown in Table 3. Although a minute amount of radioactivity appeared in the “glycogen” fraction of platelets, most of this radioactivity proved to be dialyzable and therefore not actually glycogen.
Discussion
We have investigated the possible metabolism of exogenous citrate by platelets in three different systems by measuring: 1) the “disappearance” of citrate from suspensions of platelets incubated with citrate; 2) the evolution of CO, from platelets incubated with radioactive citrate; and 3) the incorporation of radioactivity from citric acid into platelet glycogen. None of these studies gives any indication that exogenous citric acid is metabolized by platelets. No citric acid was consumed by platelets. Indeed, the amount of citric acid increased progressively during incubation. Presumably, citric acid was synthesized from oxaloacetate and acetyl CoA formed from glycogen. Moreover, no evidence was obtained that appreciable amounts of citrate carbon found their way either into C o n ,the usual product of citrate metabolism by cells, or into glycogen as might occur in gluconeogenesis. Table 2. The 14C02 Production from Platelets during Incubation in the Presence of I4C-Citrate Incubation Time (rnin.)
Purified “C-Citrate
Nonpurified W-Citrate
0 60 120
0.053 ? 0.010’ 0.060 2 0.004 0.027 t 0.009
0.134 ? 0.087’ 0.116 c 0.040 0.201 c 0.060
Mean t SE in 4 experiments expressed as per cent of total radioactivity.
Volume 19
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PLATELET CITRATb UTILIZATION
Number 5
Table 3. The Incorporation of “C-Citrate into Platelet Glycogen Purified “C-Citrate’ Incubation Time (min.) -~
0 60 120
Nondialysed Glycogen ~~
0.135 ? 0.013 0.151 2 0.023 0.155 f 0.001
Nonpurified IF-Citrate’
Dialysed Glycogen
Nondialysed Glycogen
Dialysed Glycogen
0.024 f 0.007 0.013 f 0.004 0.005 f 0.003
0.156 f 0.016 0.139 2 0.016 0.205 f 0.007
0.009 f 0.004 0.017 2 0.001 0.017 2 0.002
~
Results expressed as per cent of total radioactivity incorporated.
Our conclusions clearly differ from those previously presented by Karpatki~~.~.‘ His finding that citrate affected ATP levels of incubating platelets3 cannot be considered as evidence of citrate metabolism. Citrate has the capacity to influence metabolism in a variety of ways. It can chelate calcium, magnesium and other divalent cations. Moreover, because it is a strongly negatively charged polyanion at physiologic pH levels, it can influence intracellular pH through the Donnan membrane equilibrium effect. Karpatkin‘ found that tommercially available “C-citrate was incorporated into glycogen. Commercial preparations of radioisotopes are invariably contaminated with small amounts of other radioactive compounds. We found that citrate which was commercially available in 1978 to be no exception to this rule. Presumably, the commercially available citrate which Karpatkin employed was also contaminated with various radioactive compounds. Since the incorporation of I4C “citrate” into glycogen in Karpatkin’s studies was very modest, his findings are easily consistent with the incorporation of a contaminant. No significant incorporation of citrate into platelet glycogen was found in our investigations. We find that citrate is not metabolized by platelets. It is apparent that other sources
of metabolic energy must be made available during platelet storage. References 1. Anticoagulant citrate phosphate dextrose solution. The Pharmacopeia of the United States of America. The United States Pharmacopeial Convention, Inc. 48. Mack Publishing Company, Easton, PA, 1970. 2. Hassid, W. Z., and S. Abraham: Chemical procedures for analysis of polysaccharides. Method. Enzymol. 3:34, 1957. 3. Karpatkin, S.: Studies on human platelet glycolysis. Effect of glucose, cyanide, insulin, citrate, and agglutination and contraction on platelet glycolysis. J. Clin. Invest. 46:409, 1%7. 4. A. Charmatz, and R. M. Langer: Glycogenesis and glyconeogenesis in human platelets. Incorporation of glucose, pyruvate, and citrate into platelet glycogen: Glycogen synthetase and fructose-1,bdiphosphatase activity. J. Clin. Invest. 49: 140, 1970. 5 . Siebert, G.: Citrate and isocitrate. Determination with aconitase and isocitric dehydrogenase. I n : Methods of Enzymatic Analysis. H. U. Bergmeyer, Ed., New York, Academic Press. 1963, p. 318. 6. Slichter, S. J., and L. A. Harker: Preparation and storage of platelet concentrates. 1. Factors influencing the harvest of viable platelets from whole blood. Br. J. Haematol. M395, 1976.
-.
Constantinos Tegos, M.D., Junior Research Scientist, Hematology Department, City of Hope National Medical Center, Duarte, California 91010. Ernest Beutler, M.D., Chairman, Department of Clinical Research, Scripps Clinic & Research Foundation, La Jolla, California 92037.
