A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate

Linda Labberton, Andy T. Long, Sandra J. Gendler, Christine L. Snozek, Evi X. Stavrou, Katrin F. Nickel, Coen Maas, Stefan Blankenberg, James S. Hernandez, Thomas Renné

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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 P2Y 12 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.

Original languageEnglish (US)
Pages (from-to)369-373
Number of pages5
JournalCytometry Part B - Clinical Cytometry
Volume94
Issue number2
DOIs
StatePublished - Mar 2018

Keywords

  • biomarker
  • flow cytometry
  • platelets
  • polyphosphate

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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