Platelet Glycolysis in Platelet Storage I. The Glycolytic Enzymes C. TEGOSAND E. BEUTLER From the Deparimeni of Hemaiology. Ciiy of Hope Medical Center, Duarte, Calif.

Sixteen glymlytk enzymes were measured in e-ntlpUy leukocyte-ltn platelets before storage and daily after room temperature storage of phtekts as concentrates. Most of the glycolytk enzymes proved to be very stabk to storage. Even the most severely Pnected enzymes, enolsse, triosephoepbnte Isomerase, and lactate dehydmgenue lost less than onequarter of their enzyme activity. Depletion of glymlytlc enzymes does not acem to be a mdor factor in the storage lesion of platelets.

As TRANSFUSION OF PLATELETS has become more important in medical practice, platelet storage has received increasing attention. There is now general agreement that platelet concentrates are best stored at room temperature, and that they may be transfused for 72 hours after collection. The normal source of energy for platelets is the insulin-sensitive catabolism of glucose.* Although numerous investigations of activities of glycolytic enzymes and glucose consumption have been performed on freshly drawn platelets little is known concerning platelet glycolysis after prolonged room temperature storage. We have now initiated a series of investigations of the glycolytic pathway in stored platelets. In these studies, we have stored platelets as platelet concentrates of the type prepared in most blood banks, freed them of contaminating leukocytes a n d examined various aspects of their capacity t o metabolize glucose.

Materials and Methods Four-hundred fifty ml of blood were collected into 63 ml of citrate-phosphate-dextrosesolution. Supported in part by grant HE 07449 from the National Institutes of Health. Received for publication April 20, 1978; accepted May 23, 1978.

The blood was centrifuged for 9 minutes at between 18 and 22 C in a Sorvall RC-3 centrifuge using a HG4L rotor at 1,000 x g at the bottom of the cup (1900 rpm). The plateletrich plasma was pressed into a satellite bag which was now centrifuged for 20 minutes at 3000 x g (3400 rpm). All but approximately 35 ml of the platelet-poor plasma were pressed off and the sedimented platelets and contaminating leukocytes were resuspended. Three ml of the platelet concentrate was removed from the bag with sterile precautions, and sufficient neutralized ethylene diamine tetraacetic acid (EDTA) was added to provide a final concentration of 5 mM. The remainder of the platelet concentrate was stored at between 20 and 24 C attached to a slowly vertically rotating platform. The platelet concentrate which had been removed from the bag was diluted with nine volumes of the platelet-poor plasma and recentrifuged in a plastic centrifuge tube at 900 x g (measured at the tip) for 9 minutes at 18 to 20 C. This served to remove contaminating leukocytes while leaving at least 60 per cent of the platelets in suspension. The ratio of platelets to leukocytes in the suspension was 150,000:1. Finally, the platelets in the supernatant suspension were sedimented by centrifuging at 3,000 x g (3400 rpm) for 20 minutes, the supernatant plasma was removed, and the platelets washed two times in a solution containing 123 mM sodium chloride, 30 mM triethanolamine-HC1buffer, pH 7.8 (25 C), 1 mM potassium phosphate, 1.4 mM glucose and 5 mM EDTA. After the second washing, the sedimented platelets were resuspended in 0.4 ml of saline. Two-tenths ml of the suspension was mixed with 1.8 ml of a stabilizing solution containing 1 mM EDTA and 0.7 mM p-mercaptoethanol,' and saponin (Sigma) was added to give a concentration of 0.3 per cent. The platelet lysate was stored in ice. Phosphofructokinase assays were carried out immediately, and all of the other enzyme assays were carried out on the same day. The assay procedures were carried out as described previously for red blood cells,' except that the amount of platelet lysate used was chosen, in each case, so that there was a linear

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temperature had remarkably little effect upon the activities of most of the enzymes measured. Most severely affected were enolase, triosephosphate isomerase, and lactate dehydrogenase, but not even these lost as much as a quarter of enzyme activity.

+-

50 40

Discussion

0

1

1

2

Storopr lim

4

(Doyr)

FIG.1. The effect of storage on the activities of platelet enzymes. The values in the first three days represent the results obtained on five units of platelets. On day four, measurements were made on only four of the units. The following abbreviations are used: TPI = triosephosphatc isomerase; LDH = lactate dehydrogenase; PGK = phosphoglycerate kinase; GAPD = glyceraldehyde phosphate dehydrogenase; GPI = glucose phosphate isomerase;PK = pyruvate kinase; MPGM = monophosphoglyceromutase; GSH-Px = glutathione peroxidase; PGM = phosphoglucomutase; ENOL = enolase; HX = hexokinase; G-6-PD = glucose-&phosphate dehydrogenase; PFK = phosphofructokinase;GR = glutathione reductase; ALD = aldolase; 6PGD = dphosphogluconic dehydrogenase. relationship between volume of lysate and observed reaction of velocity. Protein estimations were carried out on an aliquot of the platelet suspension using the method of Lowry ef a1.3 and platelet enzyme activity was expressed as pmoles of substrate consumed per minute per mg of protein (I.U./mg protein).

Results Plateletsfrom five units of blood were stored for three days and daily assays of 16 enzymes were performed (Fig. 1). The activity of each enzyme at the end of storage from each of the units of platelets was divided by the prestorage enzyme activity. The mean of this ratio and its standard emor are recorded in Table 1. It is apparent that even three days' storage at room

The stability on storage appears to be analogous to the stability of red blood cell glycolytic enzymes, most of which show relatively little decline after five days of storage at room temperature. It is of interest that triosephosphate isomerase, one of the more labile of the platelet enzymes is also among the most labile enzymes in erythrocytes. Murphy and Gardnex-' found that glucose consumption and lactate production of platelets which had been stored for three to four days at room temperature in plateletrich plasma or as platelet concentrates for 24 hours actually increased at the end of 24 hours of storage. These findings are consistent with the excellent maintenance of platelet glycolytic enzymes over a threeTable 1. Ratio of Activities of Platelet Enzymes after Three Days of Storage to the Prestorage Levels. The Mean of the Ratios of 5 Units of Stored Platelets and Their Standard Error are Given

Enzyme

Activity Day 31 Activity Day 0 f 1 SE

Hx GPI PFK ALD TPI GAPD PGK MPGM ENOL PK LDH G6PD 6PGD GR GSH-PX PGM

0.913 f 0.040' 0.787 2 0.046t 0.842 f 0.047t 0.965 f 0.062 0.784 f 0.046t 0.933 f 0.048 0.892 f 0.043' 0.804 f 0.091' 0.781 f 0.060t 0.862 f 0.036t 0.779 f 0.063t 0.983 2 0.106 0.803 2 0.067' 0.915 f 0.088 0.792 f 0.1 19 0.924 f 0.092

.01 < p < .05.

t p < .01.

Abbreviations are given in legend for Figure 1.

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day storage period. Indeed, the relatively modest decline in platelet enzyme activities during storage suggest that it is unlikely that the limiting factor in storage is a glycolytic enzyme.

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and R. J. Randall: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265, 1951. 4. Murphy, S., and F. H. Gardner: Platelet storage

at 22°C. metabolic, morphologic, and functional studies. J. Clin. Invest. 50370, 1971.

References 1. Beutler, E.: Red Cell Metabolism. A Manual of

Biochemical Methods, 2nd Ed. New York, Grune & Stratton, 1975. 2. 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, 1967. 3. Lowry, 0. H., N. J. Rosebrough, A. L. Fan,

Constantinos Tegos, M.D., Attending Physician, First Department of Internal Medicine, Veterans Hospital, University of Athens. Current address: City of Hope Medical Center, 1500 E. Duarte Road, Duarte, California 91010. Ernest Beutler, M.D., Chairman, Division of Medicine, City of Hope Medical Center.

Platelet glycolysis in platelet storage I. The glycolytic enzymes.

Platelet Glycolysis in Platelet Storage I. The Glycolytic Enzymes C. TEGOSAND E. BEUTLER From the Deparimeni of Hemaiology. Ciiy of Hope Medical Cente...
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