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.
Highlights
The platelets are the paramount element of hemostasis and contribute to a variety of other normal and pathological processes, including thrombosis, inflammation, and tumor development [1,2]
We tested the applicability of our approach to study activation of single freely moving platelets induced by the addition of adenosine diphosphate (ADP)
It is well known that ADP binds to purinergic receptors on the cell surface and triggers the signaling cascade which eventually results in the opening of calcium ion channels on the intracellular stores
Summary
The platelets are the paramount element of hemostasis and contribute to a variety of other normal and pathological processes, including thrombosis, inflammation, and tumor development [1,2]. The first step of this process is platelet activation, which comprises a series of prothrombotic events, triggered by the increase of intracellular calcium [4]. It is highly anticipated that in-depth study of platelet activation and development of advanced methods for its assessment could contribute to further progress in cardiovascular medicine. It is well-known that platelet activation is triggered by a number of regulators, or agonists. These regulators, such as collagen and adenosine diphosphate (ADP), could be released from the injured vascular wall or synthesized by previously activated cells to provide positive feedback loop. Platelet activation signaling involves the sharp increase in intracellular concentration of calcium ions; the downstream effects include the platelet shape change [6], activation of the integrin family receptors, leading to platelet adhesion and aggregation [7], secretion of granules, including dense ADP-containing ones, and presentation of procoagulant factors on the platelet surface [8]
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