A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate
| Autor: | Christine L.H. Snozek, Katrin F. Nickel, Evi X. Stavrou, James S Hernandez, Thomas Renné, Linda Labberton, Sandra J. Gendler, Coen Maas, Andy T. Long, Stefan Blankenberg |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Histology medicine.diagnostic_test Activator (genetics) Polyphosphate Cell Biology 3. Good health Pathology and Forensic Medicine Flow cytometry 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology P2Y12 chemistry Biochemistry Thrombin receptor medicine Platelet Platelet activation Exopolyphosphatase |
| Zdroj: | Cytometry Part B: Clinical Cytometry. 94:369-373 |
| ISSN: | 1552-4949 |
| Popis: | Background Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. Methods and Results Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. Conclusion This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society |
| Databáze: | OpenAIRE |
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