METABOLIC PATTERNS OF PLATELETS - IMPACT ON STORAGE FOR TRANSFUSION

SCOTT MURPHY

Key words: Platelet storage, energy metabolism

The energy metabolism of platelets has been under intensive study for the past thirty years. It is known that their adenine nucleotides are partitioned into at least two pools, a storage and a metabolic pool. The storage pool which contains approximately equal amount of ADP and ATP is released when platelets are stimulated by agonists. The metabolic pool which contains predominately ATP provides not only the energy required for platelet activation but also, we assume, the energy required for maintaining the integrity of the cell during circulation in vivo and during storage for transfusion at 22OC. During optimal storage for more than one week, platelet ATP levels fall by no more that 20%[1]. Since there is considerable ATP turnover during storage[2], metabolic pathways which fuel regeneration of ATP from ADP must be active. One of them, glycolysis, the conversion of glucose to lactic acid, is very active, resulting in the accumulation of 2.5mM lactate during each day of storage of platelet concentrates (PC) prepared by the PRP methodp]. A hydrogen cation is produced with each lactate anion. Since fall in pH is deleterious for platelet viability[4], storage at 22OC would never have been successful were it not for the fortunate fact that plasma, the most widely used storage medium, contains 20mM bicarbonate which buffers the hydrogen ions sufficiently for at least one week. Any proposed synthetic storage medium must provide a mechanism to buffer the hydrogen ions produced by platelet glycolysis. Glycolysis fuels the regeneration of only one molecule of ATP for each molecule of lactate produced. The other metabolic pathway available, oxidative phosphorylation is much more efficient regenerating six molecules of ATP for each molecule of oxygen utilized. Since the rates of lactate production and oxygen consumption are equal during platelet storage[l] six of seven ATP molecules regenerated must be fueled by the oxidative pathway. This explains why platelets must be stored in a container which allows oxygen penetration adequate to meet the platelets' needs. With inadequate oxygen, the cell turns to glycolysis and increases its rate of production of lactate and hydrogen ions by as much as 7-fold, resulting in fall in pH and loss of viability[4]. The 7-fold increase would be predicted if glycolysis were required to completely compensate for a complete absence of oxidative metabolism. There are a variety of fuels available for oxidative metabolism, and different cells make different choices in different circumstances. Pyruvate which is the precursor of lactate in glycolysis, free fatty acids in plasma, and the ketone bodies such as beta-hydroxybutyrate and acetoacetate may all be converted to acetyl CoA which then condenses with oxaloacetate to form citrate as it enters the tricarboxylic acid (TCA) cycle (Figure 1). Amino acids such as glutamine and glutamate may be deaminated to form constituents of the TCA cycle such as alpha-ketoglutarate which is formed from glutamate (Figure 1). The platelet is capable of using all of these fuels to a greater or lesser extent depending on the circumstances. During storage in plasma, the most readily available fuels are pyruvate and free fatty acids. Calculating from the fact that the oxidation of one pyruvate molecule requires 2.5 oxygen molecules, all of the PC's oxygen consumption could be fueled by utilization of 1 mM of the 2.5 mM pyruvate/lactate produced daily. If this were occurring, one would predict the ratio of lactate production/glucose utilization to be 1.4/1. However, experimentally, the ratio is closer to 2/1[3] suggesting that relatively little pyruvate is oxidized and that some other fuel must be utilized. Recent evidence suggests that free fatty acids[5] and the amino acid, glutamine[6], may be utilized and that the free fatty acids are, by far, the most important quantitatively. The literature does not contain studies of the platelet's ability to metabolize ketone bodies. However, the first successful reports of platelet storage in a synthetic medium utilized a medium containing a similar molecule, acetate. It was subsequently shown[7,8] that acetate was so vigorously oxidized that it was providing all of the substrate for oxidative metabolism, presumably suppressing and replacing the utilization of free fatty

271

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Figure 1.

Metabolic pathways in platelets. The figure shows glycolysis from glucose to lactate as well as the points of entry for pyruvate, free fatty acids, ketone bodies, acetate, and the amino acid, glutamine, into the tricarboxylic acid cycle.

acids, Acetate is present in only micromolar concentrations in normal human plasma, but it can be a preferred substrate for oxidation by human tissues when supplied exogenously as, for example, in hemodialysis for renal failure. The capacity of human tissue to oxidize acetate must reflect the fact that it is a major substrate for many of our mammalian relatives such as the cow whose plasma acetate concentration is approximately 5 mM. Acetate is one of the major fuels produced for the cow by bacterial action in its rumen. The oxidation of acetate provides an unexpected benefit for platelet storage in a synthetic medium. First, the rate of production of lactic acid is suppressed by approximately 40%. Second, the metabolism of an organic anion requires that a hydrogen ion be utilized as well thus providing a buffering effect for any tendency to acidosis. A 16- or 18-carbon fatty acid takes with it only one hydrogen ion during its oxidation whereas the equivalent amount of carbon as acetate takes eight or nine hydrogen ions. Thus, acetate is not only a fuel, but it acts as a buffer as well in spite of the fact that its pk is approximately 5.0. In practice, acetate suffices as the only buffer required during platelet storage in a synthetic medium [8]. In summary, the details of platelet energy metabolism are now being elucidated. Platelet storage for transfusion continues to improve as this information is applied to clinical practice.

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References 1.

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Bertolini F., Rebulla T., Poretti L., and Murphy S: Platelet quality after 15-day storage of platelet concentrates prepared from buffy coats and stored in a glucose-free crystaloid medium. Transfusion 32:9-16, 1992. Murphy S., and Edenbrandt C: Adenine and guanine nucleotide metabolism during platelet storage at 220C. Blood 76:1884- 1892, 1990. Kilkson H., Holme S., and Murphy S: Platelet metabolism during storage of platelet concentrates at 220C. Blood 64:406-414, 1984. Murphy S: Platelet storage for transfusion. Seminars in Hematology 22:165-177, 1985. Cesar J., DiMinno G., Alam I., Silver M., and Murphy S: Plasma free fatty acid metabolism during storage of platelet concentrates for transfusion. Transfusion 27:434-437, 1987. Murphy S., Munoz S., Parry-Billings M., and Newsholme E: Amino acid metabolism during platelet storage for transfusion. British Journal of Haematology 81 :585-590, 1992. Bertolini F., Murphy S., Rebulla P., and Sirchia G: Role of acetate during platelet storage in a synthetic medium. Transfusion

Shimizu T., and Murphy S: Roles of acetate and phosphate in the successful storage of platelet concentrates prepared with an acetate-containing additive solution. Transfusion 33:304-310, 1993.

32:152-156, 1992.

SCOTT MURPHY

Cardeza Foundation for Hematologic Research Jefferson Medical College Penn-Jersey Region-American Red Cross Philadelphia, PA USA

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