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A new technique was developed for the measurement of extracellular free ATP in very small samples of whole blood using the luciferin-luciferase enzyme system. The method had a very low background corresponding to approximately 10(-16) mol ATP. ATP was measured in blood as it emerged during haemostasis following precise puncture of rat and rabbit arteries and after standardized incisions of human skin by the Simplate device. The initial concentration of free ATP in blood emerging 2-4 s after vascular injury was about 2 X 10(-7) M in rats and rabbits and about 2 X 10(-6) M in humans. The free-ATP concentration increased to 2 X 10(-5) M 3-5 min after injury and these increases could be prevented by heparin (20 u./ml). The source of the initial free ATP was identified as damaged cells in the injured vessel wall. Sufficient ADP, both released as such with ATP and generated by enzymic dephosphorylation of ATP, would be present at the site of injury to initiate haemostatic aggregation of platelets.

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