A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.
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.



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 P2Y12receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner.



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.
Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet and University Hospital, Stockholm, Sweden.|The Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Linda
L
Labberton
The Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Andy T
AT
Long
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic in Arizona, Scottsdale, Arizona.
Sandra J
SJ
Gendler
Department of Laboratory Medicine and Pathology, Mayo Clinic in Arizona, Scottsdale, Arizona.
Christine L
CL
Snozek
Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio.|Department of Medicine, Louis Stokes Veterans Administration Hospital, Cleveland, Ohio.
Evi X
EX
Stavrou
Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet and University Hospital, Stockholm, Sweden.|The Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Katrin F
KF
Nickel
Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
Coen
C
Maas
Department of General and Interventional Cardiology, University Heart Center Hamburg-Eppendorf, Hamburg, Germany.
Stefan
S
Blankenberg
Department of Laboratory Medicine and Pathology, Mayo Clinic in Arizona, Scottsdale, Arizona.
James S
JS
Hernandez
Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet and University Hospital, Stockholm, Sweden.|The Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Thomas
T
Renné
D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001777:Blood Coagulation / Q000502:physiology; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D015539:Platelet Activation / Q000502:physiology; D011122:Polyphosphates / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism / Q000502:physiology
D001426:Bacterial Proteins; D011122:Polyphosphates; D013917:Thrombin; D017766:Acid Anhydride Hydrolases; C513394:Ppx protein, Escherichia coli
biomarker; flow cytometry; platelets; polyphosphate