2. Academic Validation
  3. Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

  • Cytometry A. 2020 Sep;97(9):933-944. doi: 10.1002/cyto.a.24017.
Alessandro Aliotta 1,  Debora Bertaggia Calderara 1,  Lorenzo Alberio 1
Affiliations

Affiliation

  • 1 Hemostasis and Platelet Research Laboratory, Division of Hematology and Central Hematology Laboratory, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland.
PMID: 32338820 DOI: 10.1002/cyto.a.24017
Abstract

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow-cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real-time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo-3 (CA2+ ), ION NaTRIUM Green-2 (Na+ ), and ION Potassium Green-2 (K+ ). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, Thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (Collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry.

Keywords

blood platelets; flow cytometry; ion transport; kinetics; platelet activation.

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