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Cytometry B Clin Cytom. 2018 Mar;94(2):369-373. doi: 10.1002/cyto.b.21492. Epub 2016 Nov 28.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

Author information

1
Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet and University Hospital, Stockholm, Sweden.
2
The Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
3
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic in Arizona, Scottsdale, Arizona.
4
Department of Laboratory Medicine and Pathology, Mayo Clinic in Arizona, Scottsdale, Arizona.
5
Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio.
6
Department of Medicine, Louis Stokes Veterans Administration Hospital, Cleveland, Ohio.
7
Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
8
Department of General and Interventional Cardiology, University Heart Center Hamburg-Eppendorf, Hamburg, Germany.

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 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.

KEYWORDS:

biomarker; flow cytometry; platelets; polyphosphate

PMID:
27813345
DOI:
10.1002/cyto.b.21492

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