Metabolic energy is required in human platelets at any stage during optical aggregation and secretion.

The relationship between metabolic energy and platelet aggregation and secretion was investigated by sudden exhaustion of the cell energy content after these platelet responses had been initiated. In normal platelets, optical aggregation was at any stage susceptible to energy exhaustion, whereas single platelet disappearance and secretion were hardly affected. Prelowering the platelet energy content, while preserving the adenylate energy charge, made both optical aggregation and the secretion from dense, alpha- and acid hydrolase-containing granules susceptible to energy exhaustion, but single platelet disappearance was not affected. Complete arrest of secretion occurred when the energy content had fallen below 3-3.5 mumol ATP equivalents (ATPeq)/10(11) platelets, while optical aggregation was interrupted below 2-2.5 mumol ATPeq/10(11) platelets. At any stage of optical aggregation and the three secretion responses, the dependence on energy remained the same, indicating a tight coupling between these functions and metabolic energy, which held during the entire responses.

Balanced contribution of glycolytic and adenylate pool in supply of metabolic energy in platelets.

When platelets are treated with H2O2 the metabolic ATP content decreases sharply (Holmsen, H., and Robkin, L. (1977) J. Biol. Chem. 252, 1752-1757). Here we report that the loss of metabolic energy is fully recovered in phosphorylated glycolytic intermediates. A mixture of antimycin A/2-deoxy-D-glucose/D-gluconic acid-1,5-lactone blocks mitochondrial ATP resynthesis and prevents the entry of sugars into the glycolytic sequence. The energy-rich phosphates in the adenylate and the glycolytic pool are then consumed in a specific order. First, the glycolytic pool is consumed at a rate of 4.5 mumol of ATP equivalents/min/10(11) cells, and metabolic ATP and ADP are kept stable; then the consumption of the glycolytic pool decreases and metabolic ATP and ADP are consumed, together keeping up with the same rate of energy consumption. Thrombin stimulation increases the energy consumption to about 17 mumol of ATPeq/min/10(11) cells which is now furnished by both the glycolytic and the adenylate pool, again with a preferential consumption of the former. The results show that H2O2 triggers a shift of energy-rich phosphates from the adenylate to the glycolytic pool and that the latter remains rapidly accessible to energy consumption thereby stabilizing the level of metabolic ATP. The adenylate energy charge is independent of the distribution of energy among the two pools, which extends its importance to the regulation of energy supply and demand beyond the adenylate pool.

Comparative studies on the energetics of platelet responses induced by different agonists.

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.

Kinetics of energy consumption in human platelets with blocked ATP regeneration.

Abrupt arrest of ATP resynthesis in blood platelets induces a rapid decline in metabolic ATP-ADP. This decline is biexponential with a 7-fold difference in the rate-constants of the two components. Stimulation with thrombin increases both rate-constants, and raises the relative contribution of the rapid component from 60 to 90% of total. The initial decline can be approximated by a single exponential term, yielding the rate-constant for initial ATP hydrolysis. Since this initial decline reflects energy consumption of undisturbed platelets, this approach offers a sensitive means to determine energy consumption and ATP turnover within short time intervals.

Quantification of energy consumption in platelets during thrombin-induced aggregation and secretion. Tight coupling between platelet responses and the increment in energy consumption.

The involvement of metabolic energy in platelet responses was investigated by measuring the energy consumption during aggregation and secretion from dense, alpha- and acid-hydrolase-containing granules. Gel-filtered human platelets were stimulated with different amounts of thrombin (0.05-5.0 units X ml-1). At various stages during aggregation and secretion the energy consumption was measured from the changes in metabolic ATP and ADP following abrupt arrest of ATP resynthesis. Stimulation with 5 units of thrombin X ml-1 increased the energy consumption from 6.2 +/- 0.9 to 17.8 +/- 0.4 mumol of ATPeq. X min-1 X (10(11) platelets)-1 during the first 15 s. It decreased thereafter and returned to values found in resting cells after about 30 s. With 0.05 unit of thrombin X ml-1, the energy consumption accelerated more slowly and took at least 3 min before it normalized. A strong positive correlation was found between the velocities of the three secretion responses and the concurrent energy consumption (a) at different stages of the responses induced by a given dose of thrombin, and (b) at different secretion velocities initiated by different amounts of thrombin. When, at different stages of the responses, the extent of secretion was compared with the amount of energy that had been consumed, a strong linear correlation was found with the increment in energy consumption but not with the total energy consumption. This correlation was independent of the concentration of thrombin and indicated that complete secretion from dense, alpha- and acid-hydrolase-containing granules was paralleled by an increment of 4.0, 6.5 and 6.7 mumol of ATPeq. X (10(11) platelets)-1, respectively. An energy cost of 0.7 mumol of ATPeq. X (10(11) platelets)-1 was calculated for separate dense-granule secretion, whereas the combined alpha- and acid-hydrolase granule secretion required 5.3 mumol of ATPeq. X (10(11) platelets)-1. There was no correlation between energy consumption and optical aggregation. In contrast, the rate of single platelet disappearance, which is a measure for the early formation of small aggregates, correlated closely with the rate of energy consumption. Compared with secretion, however, the energy requirement of single platelet disappearance was minor, since 2mM-EDTA completely prevented this response but decreased the energy consumption only slightly. An increase of 0.5-1.0 mumol of ATPeq. X (10(11) platelets)-1 was seen before single platelet disappearance and the three secretion responses were initiated, indicating an increase in energy consuming processes that preceded these responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired secretion defect in platelets after cryopreservation in dimethyl sulfoxide.

Despite the use of preservatives, platelets are severely damaged during cryopreservation and, following freezing, function poorly in a number of in vitro tests. We report here that cryopreserved platelets show diminished aggregation in response to collagen. This may be a consequence of a secretion defect as evidenced by a 20 to 30 percent loss of dense- and alpha-granule content (p less than 0.05) and an impaired secretion mechanism. Analysis of adenine nucleotides confirmed the defect in dense granule adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content (storage pool), but in addition revealed a 50 percent fall in cytosolic ATP (metabolic pool). In contrast, the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate), was normal. We concluded that platelet cryopreservation leads to a secretion defect, probably as a result of activation during freezing and thawing procedures.

The energetics of early platelet responses. Energy consumption during shape change and aggregation with special reference to protein phosphorylation and the polyphosphoinositide cycle.

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.