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Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors.

ABSTRACT: Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank's balanced salt solution (HBSS) and Tyrode's buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors.

SUBMITTER: Wang L

PROVIDER: S-EPMC6786574 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors.

Wang Lili L Li Yunqian Y Guo Ran R Li Shanshan S Chang Anqi A Zhu Zhixiang Z Tu Pengfei P

PloS one 20191010 10

Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence anal ...[more]

PMID: 31600247

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Project description:BackgroundPrevious studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage.Study design and methodsThe optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference.ResultsBoth PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis.ConclusionPACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

Project description:Background and aimsStudies from our group and others demonstrate residual fluid in 42% to 95% of endoscope working channels despite high-level disinfection and drying. Additionally, persistent simethicone has been reported in endoscope channels despite reprocessing.MethodsEndoscopy was performed by using water or varied simethicone concentrations (0.5%, 1%, 3%) for flushing. After high-level disinfection/drying, we inspected endoscope working channels for retained fluid by using the SteriCam borescope. Working channel rinsates were evaluated for adenosine triphosphate (ATP) bioluminescence. Fourier transform infrared spectroscopy was performed on fluid droplets gathered from a colonoscope in which low-concentration simethicone was used.ResultsUse of medium/high concentrations of simethicone resulted in a higher mean number of fluid droplets (13.5/17.3 droplets, respectively) and ATP bioluminescence values (20.6/23 relative light units [RLUs], respectively) compared with that of procedures using only water (6.3 droplets/10.9 RLUs; P < .001). Two automated endoscope reprocessing cycles resulted in return of a fluid droplet and ATP bioluminescence values to ranges similar to that of procedures that used only water (P = .56). Low-concentration simethicone did not increase the mean residual fluid or ATP bioluminescence values compared with procedures that used only water (5.8 droplets/15.6 RLUs). Fourier transform infrared analysis revealed simethicone in the endoscope working channel after use of low-concentration simethicone.ConclusionsUse of medium/high concentrations of simethicone is associated with retention of increased fluid droplets and higher ATP bioluminescence values in endoscope working channels, compared with endoscopes in which water or low concentration simethicone was used. However, simethicone is detectable in endoscopes despite reprocessing, even when it is utilized in low concentrations. Our data suggest that when simethicone is used, it should be used in the lowest concentration possible. Facilities may consider 2 automated endoscope reprocessor cycles for reprocessing of endoscopes when simethicone has been used.

Project description:BackgroundNutritional supplements designed to increase adenosine 5'-triphosphate (ATP) concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity exercise. Oral ATP supplements have beneficial effects in some but not all studies examining physical performance. One of the remaining questions is whether orally administered ATP is bioavailable. We investigated whether acute supplementation with oral ATP administered as enteric-coated pellets led to increased concentrations of ATP or its metabolites in the circulation.MethodsEight healthy volunteers participated in a cross-over study. Participants were given in random order single doses of 5000?mg ATP or placebo. To prevent degradation of ATP in the acidic environment of the stomach, the supplement was administered via two types of pH-sensitive, enteric-coated pellets (targeted at release in the proximal or distal small intestine), or via a naso-duodenal tube. Blood ATP and metabolite concentrations were monitored by HPLC for 4.5?h (naso-duodenal tube) or 7?h (pellets) post-administration. Areas under the concentration vs. time curve were calculated and compared by paired-samples t-tests.ResultsATP concentrations in blood did not increase after ATP supplementation via enteric-coated pellets or naso-duodenal tube. In contrast, concentrations of the final catabolic product of ATP, uric acid, were significantly increased compared to placebo by ~50% after administration via proximal-release pellets (P?=?0.003) and naso-duodenal tube (P?=?0.001), but not after administration via distal-release pellets.ConclusionsA single dose of orally administered ATP is not bioavailable, and this may explain why several studies did not find ergogenic effects of oral ATP supplementation. On the other hand, increases in uric acid after release of ATP in the proximal part of the small intestine suggest that ATP or one of its metabolites is absorbed and metabolized. Uric acid itself may have ergogenic effects, but this needs further study. Also, more studies are needed to determine whether chronic administration of ATP will enhance its oral bioavailability.

Dataset Information

Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors.

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Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors.

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