Platelet Activation and Cytosolic Free Potassium Kinetics: A Dynamic Flow Cytometric Monitoring

Background and Objectives: Regulation of cytosolic ion homeostasis is an important cellular process and imbalances of intracellular ions play a role for signal transduction. Gradient of potassium (K⁺) ions are involved in cellular function and ion homeostasis. Some studies revealed that rise of cytosolic Ca²⁺ during platelet (PLT) activation triggers Ca²⁺-dependent K⁺-channels, having an impact on cytosolic K⁺ concentrations and activation endpoints. To investigate K⁺ kinetics during PLT response to agonists, we set up a continuous flow cytometric monitoring of intracellular K⁺ ion fluxes and we followed fluxes upon PLT activation.

Methods: PLTs were loaded with fluorescent potassium ion indicators Asante Potassium Green-2 (APG2) AM. After measurement of a stable baseline, PLTs were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (collagen receptor GPVI agonist) or combination thereof, in both Ca²⁺-free and Ca²⁺-supplemented buffers. Then, APG2 fluorescence was continuously acquired over time, up to 10 minutes, on an Accuri C6 flow cytometer.

Results: Optimal APG2 loading conditions (incubation time and final concentration) were set up and calibration procedure was performed with different K⁺ concentration buffers. Greater K⁺ efflux was observed in Ca²⁺-supplemented buffer compared to Ca²⁺-free buffer. Very strong and sustained K⁺ efflux was observed by combined activation with convulxin and thrombin (-120 mM). Convulxin and thrombin alone were also able to induce an important K⁺ efflux (-107 mM and -92 mM, respectively). Both selective PAR agonists, TRAP6 and AYPGKF, demonstrated also a declined intracellular K⁺ (-85 mM) but with a slower efflux kinetics. However, their combined action could replicate thrombin effect. Finally, even though a moderate K⁺ efflux was observed with ADP and U46619 alone (-76 mM and -68 mM, respectively), these agonists impacted convulxin or thrombin responses when employed in combination.

Conclusions: The present work highlights the use of K⁺ fluorescent dye for continuous flow cytometric ion monitoring. The presented data demonstrate characteristic K⁺ kinetics following PLT activation with various agonists and a Ca²⁺ dependent efflux could be demonstrated. As K⁺ ions are implicated in several cellular processes as cell shrinkage, membrane potential, calcium signaling, and even apoptosis this technique will could improve analysis of PLT signaling and may be useful for deeper investigations on PLT function and pathophysiology.

 

MissingLinkText

No conflict of interest has been declared by the author(s).