On calcium fluorophore’s impact in platelet signaling studies

Observation of calcium signaling in platelets - blood cells designed to be involved in stopping bleeding and forming blood clots - is an important part of fundamental research in hemostasis. However, such a study is possible only with the use of calcium fluorophores - small molecules that penetrate the platelet membrane due to their hydrophobic -AM part, which is then hydrolyzed by cytosol esterases. In this work, we consider the phenomenon of inhomogeneous loading of calcium fluorophores into platelets.

We used platelets from healthy adult donors loaded with various fluorescent probes (CalBryte590, DiOC6 (3), Fura Red, Fluo-4 and CellTracker Violet BMQC) and immobilized on antibodies to CD31 in parallel plane flow chambers. Total internal reflection fluorescence (TIRF) microscopy was used for observations.

We demonstrated that all studied probes are loaded heterogeneously, with 30% platelets being loaded with a probe 2-6 times higher than the population median value. Using the CalBryte590 probe as an example, we have shown that a decrease in the incubation temperature, the addition of Pluronic 127 to the incubation medium, or membrane cholesterol depletion significantly reduces the heterogeneity of the probe distribution in the population. By looking at platelet activation from the surface, we have shown that the probability of experiencing strong activation, as measured by the intensity of calcium oscillations, correlates with the amount of probe in the platelet.

Thus, we conclude that the type of fluorophore used and the conditions of its loading into platelets can significantly affect the results of experiments on the observation of calcium signaling in platelets.

Overall scheme of the assay. First, whole pre-processed blood is perfused through the flow chamber for 2.5 minutes. Then Tyrode's buffer is perfused through the chamber for 5 minutes to wash away unattached cells. Then, depending on the protocol, the microscopy video is recorded for 5-10 minutes.
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#platelets#calcium fluorophores#membrane#fluorescent probes#intracellular signaling

Presence of PI-rich vesicles is required for the PLC ζ activation according to mathematical modeling

Phospholipase C ζ (PLCζ) is an enzyme found in the cytoplasm and acrosome of mammalian spermatozoa. It catalyzes the reaction of phosphatidylinositol-4,5-phosphate hydrolysis into inositol-3-phosphate and diacylglycerol. PLCζ is present in the sperm cell acrosome and cytosol but doesn’t significantly affect its metabolism. However, after the fusion of sperm and egg membranes, its activity increases as it begins to bind membranes of the egg. It is unknown why PLCζ is inactive in spermatozoa or any type of somatic cell.

In this work, the modeling approach explains the reasons for the absence of PLCζ activity in any type of mammalian cells but eggs. A model describing the activity of PLCζ in physiological calcium concentrations was developed. It was shown that the presence of phosphoinositide-rich vesicles is required for the PLC ζ activity in mature mammalian eggs.

Scheme of the full model. (A) Reactions in the oocyte, PLCζ – calcium-free PLCζ , PLCζ_Ca – PLCζ, bound with one calcium ion, PLCζ_2Ca – PLCζ, bound with two calcium ions, PLCζ_3Ca  – with three, PLCζ_4Ca  – with four. PLCζ_m  – PLCζ, bound with cell membrane, PLCζ_(Ca m ) – PLCζ, bound with the cell membrane and one calcium ion, PLCζ_(2Ca m )– PLCζ, bound with the cell membrane and two calcium ions, PLCζ_(3Ca m ) – with the cell membrane and three calcium ions, PLCζ_(4Ca m ) – with the cell membrane and four calcium ions. PIP2 and PIP2_v  – are phosphatidylinositol-4,5 – bis-phosphates on cell and vesicle membrane accordingly. DAG and DAG_v  – diacylglycerol on cell membrane and vesicles accordingly. IP3 – inositol-3-phosphate. PLCζ_v – PLCζ, bound with vesicles, PLCζ_(Ca v ) – PLCζ, bound with vesicles and one calcium ion, PLCζ_(2Ca v ) – PLCζ, bound with vesicles and two calcium ions, PLCζ_(3Ca v ) – with vesicles and three calcium ions, PLCζ_(4Ca v ) – with vesicles and four calcium ions. (B) The sperm cell model is identical to the oocyte model except for the absence of the vesicles.
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#phospholipase Cz#calcium signaling#spermatozoa#oocyte

Patch-clamp technique for studying ion channels in activated platelets

In this study, we have revisited the existing and suggested new approaches to the use of patch-clamp methodology for measuring the activity of single ion channels and membrane potential of human platelets in the cell-attached configuration. We recorded single-channel events of platelets in the cell-attached configuration after activation with potent agonists: thrombin and ionomycin. The feasibility of platelet membrane potential measurement in cell-attached mode was investigated both experimentally and with the help of simple electrical circuits, revealing that the single-channel events can alter the recorded potential by invoking oscillations. Here, the simple approach to obtain inside-out configuration was described and calcium-dependent single-channel ion currents were recorded. Taken together, this study introduces new approaches for further investigations of the role of ion channels and membrane potential in platelet physiological and pathophysiological response.
Schematic illustration of recording currents of single ion channels of platelets during activation, excluding K<sub>v</sub> 1.3
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#platelets#patch-clamp#membrane potential#ion channel
Systems Biology and Systems Physiology: regulation of biological networks
The meeting is dedicated to the 75th anniversary of Fazly Ataullakhanov
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Illuminating the nature of complex systems