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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.

SUBMITTER: von Papen M

PROVIDER: S-EPMC3638993 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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

von Papen Michael M Gambaryan Stepan S Schütz Claudia C Geiger Jörg J

Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie 20130315 2

<h4>Background</h4>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.<h4>Methods</h4>Supernatants of washed ...[more]

PMID: 23652982

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Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Dataset Information

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

Publications

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

Similar Datasets

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