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

Abstract

The involvement of metabolic energy in platelet responses was investigated by measuring the energy consumption during aggregation and secretion from dense, α- and acid-hydrolase-containing granules. Gel-filtered human platelets were stimulated with different amounts of thrombin (0.05-5.0 units.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.ml-1 increased the energy consumption from 6.2 ± 0.9 to 17.8 ± 0.4 μmol of ATP(eq.).min-1.(1011 platelets)-1 during the first 15s. It decreased thereafter and returned to values found in resting cells after about 30s. Within 0.05 unit of thrombin.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, α- and acid-hydrolase-containing granules was paralled by an increment of 4.0, 6.5 and 6.7 μmol of ATP(eq.).(1011 platelets)-1, respectively. An energy cost of 0.7 μmol of ATP(eq.).(1011 platelets)-1 was calculated for separate dense-granule secretion, whereas the combined α- and acid-hydrolase granule secretion required 5.3 μmol of ATP(eq.).(1011 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 μmol of ATP(eq.).(1011 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. The current studies indicate that, throughout the responses, secretion is tightly coupled to the increment in energy consumption. The association with energy metabolism is much weaker for aggregation.