Assessment Of Energy Costs Of Secretory Responses In Platelets By Abrupt Arrest Of ATP Regeneration
Thrombosis and Haemostasis
A new method has been developed for the quantitative assessment of energy consuming processes in platelets. Under carefully controlled metabolic conditions ATP resynthesis is abruptly blocked by a cocktail of metabolic inhibitors. This leads to a fall in metabolic ATP, which is linear with time between 0 and 30 sec after addition of the inhibitors. Evidence is presented that this fall reflects the velocity by which the platelets consume metabolic energy prior to addition of the inhibitors.
... he inhibitors. Resting platelets consume 4 μmol ATP equivalents/min/1011 cells at 37° and 0.5 μmol (same units) at 15°C. When thrombin (5 U/ml) is included in the inhibitor-mixture, aggregation and secretion of dense granules (3H-serotonin), α-granules (β-thromboglobulin) and lysosomal granules (N acetyl β glucosaminidase) follow despite the arrest in ATP resynthesis. The fall in metabolic ATP is now much steeper, reflecting an increase in energy consumption during these functions. Using changes in temperature as a means to affect secretion and energy metabolism, secretion velocity (measured between 0 and 10 sec after thrombin addition) can be compared with simultaneous energy consumption (measured between 0 and 30 sec after thrombin addition). At a consumption of 12 ymol ATP/min/1011 cells secretion velocity of dense-, α- and lysosomal granules is 100, 95 and 50% of uninhibited suspensions, respectively. At 6 μmol (same units) these percentages are 70, 35 and 25%.If thrombin is added after addition of the inhibitors thereby initiating secretion at lowered metabolic ATP levels, secretion is slower as metabolic ATP is lower. Again lysosomal granule secretion is more inhibited than α-granule secretioiy. which is slower than dense granule secretion. These data reflect an increasing need for metabolic energy in the order: dense-, α- and lysosomal granule secretion.