The bioluminescent detection of platelet released ATP: collagen-induced release and potential errors

The bioluminescent detection of ATP released from activated platelets is an important diagnostic and experimental assay. Potential errors in the interpretation of the data may be introduced due to the lability of luciferin-luciferase and the amount of platelet agonist employed. Loss of luciferin-luciferase activity is temperature dependent with a 50% decrease in activity in 1-4 min at 37 degrees C. Plasma components do not appear to contribute to the inactivation of the detection system. Due to the significant loss of enzyme activity at variable times, the method of standardizing ATP concentrations is crucial for the accurate determination of ATP released from activated platelets. A nearly 5-fold error is introduced into the routinely employed assay procedure where the standard ATP concentration is determined 5 min after the addition of agonist. This report demonstrates that the standard ATP concentration must be determined with a separate platelet sample at the same time as the ATP was released from the agonist-induced experimental platelet sample. A second significant error in the assay system may be introduced by the agonist concentration employed even when the final level of aggregation is the same. When collagen is employed as the agonist the amount of ATP released appears to depend, in part, on the initial intensity of the aggregation response and not on collagen type (Type I versus IV). The corrective procedures described here for the detection of ATP are not likely to change the qualitative results of most studies but would significantly alter the quantitative results.