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Optical uncaging of ADP reveals the early calcium dynamics in single, freely moving platelets - PubMed

  • ️Wed Jan 01 2020

. 2020 May 27;11(6):3319-3330.

doi: 10.1364/BOE.392745. eCollection 2020 Jun 1.

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Optical uncaging of ADP reveals the early calcium dynamics in single, freely moving platelets

Darya V Spiryova et al. Biomed Opt Express. 2020.

Abstract

Platelet activation is considered to be a cornerstone in pathogenesis of cardiovascular disease. The assessment of platelet activation at the single-cell level is a promising approach for the research of platelet function in physiological and pathological conditions. Previous studies used the immobilization of platelets on the surface, which significantly alters the activation signaling. Here we show that the use of photolabile "caged" analog of ADP allows one to track the very early stage of platelet activation in single, freely moving cells. In this approach, the diffusion step and ADP receptor ligation are separated in time, and a millisecond-timescale optical pulse may trigger the activation. The technique allows us to measure the delay (lag time) between the stimulus and calcium response in platelets. We also propose a simple model function for calcium peaks, which is in good agreement with the measured data. The proposed technique and model function can be used for in-depth studies of platelet physiology.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.

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Conflict of interest statement

All authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.

Schematic layout of experimental setup for optical activation of platelets. Inverted microscope (Carl Zeiss AxioVert.A1) is used to monitor the level of intracellular calcium during platelets activation. The activation is caused by “uncaging” the cagedADP upon the UV irradiation.

Fig. 2.
Fig. 2.

Outline of experiments and tracking procedure. A. Typical fiald of view showing ∼100 platelets. B. An enlarged region with several platelets before the addition of ADP. One spread cell is shown (marked by yellow circle), as well as several resting platelets. C. Platelets after the addition of ADP. Activation of freely moving platelets is evident by the increase of Fluo-4 fluorescence intensity. E. The track of one cell (marked by light-blue circle) showing its trajectory and the fluorescence intensity (as point color; increases from black to green).

Fig. 3.
Fig. 3.

A Snapshots with activation of the spread cell and the graph showing the dependence of fluorescence intensity on time before and after the addition of ADP (yellow line). B. Snapshots of the typical freely moving platelet and the graph showing the dependence of fluorescence intensity on time. There are numerous calcium transients. In addition, the results of our algorithm, which seeks for calcium peaks and fit them with simple model equation, are shown by green lines. The parameters are presented in the corresponding table for each found peak.

Fig. 4.
Fig. 4.

A. Control experiments showing that two UV flashes do not influence platelet calcium signaling. Note two spontaneously activated cells which show the signaling completely independent on UV. B Several typical signals obtained during experiments with caged ADP. There are numerous calcium transients after the UV irradiation pulse (violet line) which cleaves the caged ADP.. C,D: Snapshots with activation of two different typical cells and the graph showing dependence of fluorescence intensity on time. The processing (fitting) results are shown by green line.

Fig. 5.
Fig. 5.

A,B. The histograms of the decay constant for each peak measured in ADP and caged ADP experiments. C,D. The histograms of the peak-to-peak interval obtained in ADP and caged ADP experiments. E. The histogram of appearance time (t0) for all peaks in experiment with caged ADP.

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