Determination of ATP and ADP Secretion from Human and Mouse Platelets by an HPLC Assay

Abstract

Background: Secretion of ADP and ATP is an essential prerequisite for platelet aggregation. Impaired nucleotide secretion can cause aggregation defects and increased bleeding risk. Quantitative determination of platelet nucleotide content and exocytosis is thus of importance for the characterization and diagnosis of bleeding phenotypes. For transgenic animal models with hemostatic defects analysis of potential secretion defects is as well imperative. Methods: Supernatants of washed platelets and platelet-rich plasma were analyzed by HPLC for ADP and ATP concentration. Calibration of the HPLC data was accomplished with an internal standard compensating for loss of analyte, detection sensitivity, and interference of the biomatrix. Results: HPLC analysis of nucleotide secretion was carried out with human and mouse platelets. Detection limits were determined for washed platelet and platelet-rich plasma samples. In the physiological concentration range linearity with respect to the peak area is maintained. Conclusion: The method combines reasonable sensitivity with robustness. The internal standard ensures reliable quantification of nucleotide concentrations even in presence of otherwise interfering substances. The low sample consumption renders possible the application to analysis of small samples like in mouse experiments.