Hydrogen peroxide lowers ATP levels in platelets without altering adenyalte energy charge and platelet function.

H2O2 irreversibly reduced metabolic platelet ATP levels with a corresponding accumulation of hypoxanthine. This process was enhanced by sodium azide or potassium cyanide and by increasing H2O2 concentrations. The adenylate energy charge was unaltered when less than two thirds of the metabolic ATP had disappeared but decreased markedly when more ATP disappeared. Platelet shape change, primary aggregation, dense granule and alpha-granule secretion were unaffected by H2O2-induced lowering of ATP provided that the adenylate energy charge did not fall by more than 5%; at greater adenylate energy charge reduction, platelet functions were inhibited. These results indicate that cell functions depend more on adenyalte energy charge than on the ATP level and expands the applicability of this view from bacterial systems to a mammalian cell, the human platelet.

, and a qualitatively similar pattern was obtained with suspensions of gel-filtered platelets. The extent of this conversion was, however, greater with gel-filtered platelets than in platelet-rich plasma ( Table  I, Fig. 2). The catalase inhibitors cyanide and azide markedly increased both the rate and the extent of the H,O,-induced reduction in the ['QATP level. Incubation of gel-filtered platelets or platelet-rich plasma with azide alone did not cause any changes in the ATP level or in the adenylate energy charge (Table I). In contrast, cyanide caused a slight, slow decrease in the ATP level of platelets in both systems, especially in gel-filtered platelets, but had no noticeable effect on the adenylate energy charge (Figs. 1 and 5, Table I).
Maximal effect of H,O, on platelet YC]ATP was observed with 0.1 mM azide or 1 mM cyanide both in platelet-rich plasma and in gel-filtered platelets ( In the presence of HZ02 the radioactivity of ADP and AMP decreased almost in parallel to that of ATP. H,O,-The above results showed that H,O, affects the metabolic pool of platelet adenine nucleotides, but gave no information regarding possible effects on the storage pool. The specific radioactivity of the ATP and ADP which disappeared during incubation of platelet-rich plasma with H,02 was found to be identical with that of the ethanol-insoluble ADP fraction of the metabolic pool (Table II). Thus, H,O, causes breakdown of only the metabolic adenine nucleotides and not of the nonmetabolic (storage) pool. That H,O, did not affect the storage pool ATP and ADP in platelets can be seen directly in experiments where thrombin caused the secretion of the same amounts of storage (nonmetabolic) ATP and ADP from H,Oz-treated platelets as from control platelets (see Table IV 0 5,196 5,196 peared moved from the systems (Fig. 3) ity of platelets (and plasma) is of special interest since it has recently been shown that platelets may be exposed in vivo to H,O, in concentrations comparable to those used here (8).
Effects ofH202 on Platelet Lactate, Glycogen, and Inorganic Phosphate -The level of inorganic phosphate did either not change or decreased slightly in glucose-free suspensions of gelfiltered platelets during H,O,-induced ATP breakdown.
A pronounced increase in glycogen breakdown took place during ATP breakdown but the rate of glycogen disappearance was the same as in control platelets after the ATP level had stabilized. The increase in glycogen breakdown was not associated with a sustained increase in the lactate production (Table  III). Usually there was a small increase in lactate production during the first 6 min of incubation of platelets with H,O,; after this period lactate was produced at the same rate as in control platelets incubated with cyanide alone.
These results suggest that the H,O,-induced ATP disappearance did not take place by simple hydrolysis. It is tempting to speculate that ATP may be consumed in phosphorylation processes, possibly of glycolytic intermediates, since the increased glycogen breakdown occurred without an increase in lactate production when cyanide was present. It has been shown that perfusion of rat liver with substrates for glutathione peroxidase (H202, t-butyl, and cumene hydroperoxides) increases glycogenolysis (27) and causes lowering of the adenylate pool, however, with a lowering of the adenylate energy charge (28). Platelets contain large amounts of glutathione, glutathione peroxidase, and glutathione reductase (291, which makes it possible that there is a link between H,Oz removal, glycogen degradation, and reduction in the steady state levels of metabolic ATP. The transient fall in adenylate energy charge platelet aggregation varied much among platelet-rich plasma from different donors. We have observed the same and have also seen that the H,Oz-removing capacity (without catalase inhibitor) varied much. However, the variation in inhibitory effect and removing capacity was diminished when catalase inhibitors were used, indicating that the differences seen were partly due to variations in the catalase level. Azide was found to be a powerful inhibitor of platelet aggregation induced by ADP, epinephrine, or collagen and could not be used in the aggregation studies. Cyanide, on the other hand, had only a small inhibitory effect on aggregation (see KCN control,Fig. 41 and was used in the subsequent studies. When ADP-and epinephrine-induced platelet aggregation was measured in platelet-rich plasma following the addition of HZ09, primary aggregation was inhibited only during the period when the level of metabolic ATP was falling and the adenylate energy charge was lower than normal. As the level of ATP stabilized and the energy charge returned to normal, the rate and extent of primary platelet aggregation also returned to normal. Fig. 4 shows a typical experiment with ADP as the aggregating agent in which the variation in aggregation, [14C]adenine metabolites, and adenylate energy charge ATP Leuels, Adenylate Energy Charge, and Platelet Function are shown separately for the control (KCN) and the experimental sample (KCN + H,O,). Fig. 5 shows a typical experiment with epinephrine where the aggregation rate is given in percentage of control. When various concentrations (0.5, 1, 2, and 5 PM) of ADP or epinephrine were used, the inhibitory period lasted longer for the lower concentrations than for the higher, but when the adenylate energy charge had returned to less than 5% below the control value, no effect on aggregation was seen with any concentration of ADP or epinephrine.
The effect of H,O, on the platelet shape change process was studied with 0.2 to 2 PM ADP (in the presence of 5 mM EDTA) in platelet-rich plasma and with an initial concentration of 306 PM H,O, over a 20-min incubation period. No change in the shape change response was noted during incubation, even at an adenylate energy charge as low as 0.82 (data not shown).
Collagen-induced secretion of [14C]serotonin previously incorporated by platelets in plasma was not inhibited when the adenylate energy charge was less than 5% below control values.  whereas cY-granule secretion was inhibited at slightly reduced adenylate energy charge, but then was unaffected when the charge had returned to control values (Table IV).
Secretion and secondary aggregation in platelet-rich plasma in response to ADP and epinephrine was, however, abolished by H,O, even when the adenylate energy charge had returned to normal values.
Concluding Remarks -Since cyanide was used in studies on platelet function, oxidative regeneration of ATP did not take place and lactate production could be used as a measure of ATP turnover. Our experiments show that H,O, can be used as a tool to specifically reduce the ATP level in platelets without altering ATP turnover and adenylate energy charge. In addition, the agent is rapidly removed by the cells. Thus, at normal ATP turnover rates shape change, aggregation, dense granule secretion, and a-granule secretion are unaltered by large reductions (up to 60%) in the level of metabolic ATP, provided that the adenylate energy charge is not lowered by more than 5%. When the energy charge is lowered by more than 5% several of these platelet functions are inhibited. Evidently, these platelet functions depend more on the adenylate energy charge than on ATP concentration.
Our results strongly support the view of Atkinson (30) that the adenylate energy charge rather than [ATPI is the important regulatory parameter for control of cell functions. This has previously been demonstrated for bacteria, and the present results expand the applicability of this view to a mammalian cell, the human platelet.