A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate
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A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate. / Labberton, Linda; Long, Andy T; Gendler, Sandra J; Snozek, Christine L; Stavrou, Evi X; Nickel, Katrin F; Maas, Coen; Blankenberg, Stefan; Hernandez, James S; Renné, Thomas.
In: CYTOM PART B-CLIN CY, Vol. 94, No. 2, 03.2018, p. 369-373.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate
AU - Labberton, Linda
AU - Long, Andy T
AU - Gendler, Sandra J
AU - Snozek, Christine L
AU - Stavrou, Evi X
AU - Nickel, Katrin F
AU - Maas, Coen
AU - Blankenberg, Stefan
AU - Hernandez, James S
AU - Renné, Thomas
N1 - © 2016 International Clinical Cytometry Society.
PY - 2018/3
Y1 - 2018/3
N2 - 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 P2Y12receptor 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.
AB - 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 P2Y12receptor 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.
KW - Journal Article
U2 - 10.1002/cyto.b.21492
DO - 10.1002/cyto.b.21492
M3 - SCORING: Journal article
C2 - 27813345
VL - 94
SP - 369
EP - 373
JO - CYTOM PART B-CLIN CY
JF - CYTOM PART B-CLIN CY
SN - 1552-4949
IS - 2
ER -
