On calcium fluorophore’s impact in platelet signaling studies

Blood is an important component of the human body, however, a system for maintaining its integrity is needed - a hemostasis system, roughly divided into plasma and vascular-platelet links

[

1

Hemostasis and thrombosis beyond biochemistry: roles of geometry, flow and diffusion

M. Panteleev, N. Dashkevich, F. Ataullakhanov

Thrombosis Research. 2015, 136, 699-711

https://doi.org/10.1016/j.thromres.2015.07.025

Article | Google Scholar

]

. Platelets, small non-nuclear blood cells, are designed to stop bleeding when a vessel is damaged by forming an aggregate and providing its surface to accelerate the work of the plasma hemostasis link

[

2

Platelets and hemostasis

Panteleev M.A., Sveshnikova A.N.

Oncohematology. 2014, 9(2), 65-73

https://doi.org/10.17650/1818-8346-2014-9-2-65-73

Article | Google Scholar

]

. To accomplish this task, platelets enter the so-called activated state. When activated, platelets adhere to the surface and aggregate with each other

[

3

Platelet Integrin αIIbβ3: Mechanisms of Activation and Clustering; Involvement into the Formation of the Thrombus Heterogeneous Structure

V. Kaneva, A. Martyanov, D. Morozova, M. Panteleev, A. Sveshnikova

Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology. 2019, 13, 97-110

https://doi.org/10.1134/S1990747819010033

Article | Google Scholar

]

, change their shape, secrete the contents of granules

[

4

Platelet functional responses and signalling: the molecular relationship. Part 1: responses.

A. Sveshnikova, M. Stepanyan, M. Panteleev

Systems Biology and Physiology Reports. 2021, 1, 20-28

https://doi.org/10.52455/sbpr.01.202101014

Article | Google Scholar

]

and, in some cases, become procoagulant

[

5,

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

N. Topalov, Y. Kotova, S. Vasil’ev, M. Panteleev

British Journal of Haematology. 2012, 157, 105-115

https://doi.org/10.1111/j.1365-2141.2011.09021.x

Article | Google Scholar

6

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting

N. Podoplelova, A. Sveshnikova, Y. Kotova, A. Eckly, N. Receveur, D. Nechipurenko, S. Obydennyi, I. Kireev, C. Gachet, F. Ataullakhanov, P. Mangin, M. Panteleev

Blood. 2016, 128, 1745-1755

https://doi.org/10.1182/blood-2016-02-696898

Article | Google Scholar

]

. Eventually, a blood clot forms

[

7

Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface

D. Nechipurenko, N. Receveur, A. Yakimenko, T. Shepelyuk, A. Yakusheva, R. Kerimov, S. Obydennyy, A. Eckly, C. Léon, C. Gachet, E. Grishchuk, F. Ataullakhanov, P. Mangin, M. Panteleev

Arteriosclerosis, Thrombosis, and Vascular Biology. 2019, 39, 37-47

https://doi.org/10.1161/ATVBAHA.118.311390

Article | Google Scholar

]

.

A feature of platelet activation is its quick reaction to damage - with characteristic times of the order of one second

[

8

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets

M. HARPER, A. POOLE

Journal of Thrombosis and Haemostasis. 2011, 9, 1599-1607

https://doi.org/10.1111/j.1538-7836.2011.04393.x

Article | Google Scholar

]

. Probably, the platelet intracellular signaling system provides such a response rate due to calcium ions. In resting cells, the concentration of free calcium in the cytosol is maintained at a lower level (approximately 10-20 nM)

[

9

Heterogeneity of Integrin αIIbβ3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis

A. Martyanov, D. Morozova, M. Sorokina, A. Filkova, D. Fedorova, S. Uzueva, E. Suntsova, G. Novichkova, P. Zharkov, M. Panteleev, A. Sveshnikova

International Journal of Molecular Sciences. 2020, 21, 3035

https://doi.org/10.3390/ijms21093035

Article | Google Scholar

]

than in blood plasma (1-2 mM), and in the endoplasmic reticulum, which is considered the main store of calcium in the platelet, its concentration is about 100 - 800 μM

[

10

Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N

S. SAGE, N. PUGH, M. MASON, A. HARPER

Journal of Thrombosis and Haemostasis. 2011, 9, 540-551

https://doi.org/10.1111/j.1538-7836.2010.04159.x

Article | Google Scholar

]

. Platelet activation, like many other cells, is controlled by the concentration of calcium ions in the cytosol due to the fact that calcium ions are cofactors for many enzymes mediating platelet activation

[

11,

Platelet functional responses and signalling: the molecular relationship. Part 2: receptors.

A. Martyanov, M. Panteleev

Systems Biology and Physiology Reports. 2021, 1, 13-30

https://doi.org/10.52455/sbpr.01.202103013

Article | Google Scholar

12,

Why Calcium? How Calcium Became the Best Communicator

E. Carafoli, J. Krebs

Journal of Biological Chemistry. 2016, 291, 20849-20857

https://doi.org/10.1074/jbc.R116.735894

Article | Google Scholar

13

The calcium-signalling saga: tap water and protein crystals

E. Carafoli

Nature Reviews Molecular Cell Biology. 2003, 4, 326-332

https://doi.org/10.1038/nrm1073

Article | Google Scholar

]

.

As such an important component of platelet activation, calcium signaling has been studied for a long time both in cell suspension

[

14,

Transient Receptor Potential Channels Function as a Coincidence Signal Detector Mediating Phosphatidylserine Exposure

M. Harper, J. Londoño, K. Quick, J. Londoño, V. Flockerzi, S. Philipp, L. Birnbaumer, M. Freichel, A. Poole

Science Signaling. 2013, 6, None

https://doi.org/10.1126/scisignal.2003701

Article | Google Scholar

15

Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling

A. Sveshnikova, A. Balatskiy, A. Demianova, T. Shepelyuk, S. Shakhidzhanov, M. Balatskaya, A. Pichugin, F. Ataullakhanov, M. Panteleev

Journal of Thrombosis and Haemostasis. 2016, 14, 2045-2057

https://doi.org/10.1111/jth.13442

Article | Google Scholar

]

and in single platelets

[

16,

Roles of phospholipase C and Ca(2+)-ATPase in calcium responses of single, fibrinogen-bound platelets.

J. Heemskerk, P. Vis, M. Feijge, J. Hoyland, W. Mason, S. Sage

Journal of Biological Chemistry. 1993, 268, 356-363

https://doi.org/10.1016/S0021-9258(18)54158-2

Article | Google Scholar

17,

Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets

J. Heemskerk, J. Hoyland, W. Mason, S. Sage

Biochemical Journal. 1992, 283, 379-383

https://doi.org/10.1042/bj2830379

Article | Google Scholar

18

Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation

S. Obydennyy, A. Sveshnikova, F. Ataullakhanov, M. Panteleev

Journal of Thrombosis and Haemostasis. 2016, 14, 1867-1881

https://doi.org/10.1111/jth.13395

Article | Google Scholar

]

. The study of calcium signaling is carried out using intracellular fluorescent probes - small organic molecules-fluorophores, the parameters of excitation and / or fluorescence of which significantly change when binding calcium ions

[

19

Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

T. Ha, P. Tinnefeld

Annual Review of Physical Chemistry. 2012, 63, 595-617

https://doi.org/10.1146/annurev-physchem-032210-103340

Article | Google Scholar

]

. However, without damaging the cell membrane, fluorescent probes can be introduced into it only by diffusion, and for this the molecules are modified by the addition of an acetomethyl residue, which is hydrolyzed inside the cell by nonspecific esterases

[

20

Measurement of Intracellular Calcium

A. Takahashi, P. Camacho, J. Lechleiter, B. Herman

Physiological Reviews. 1999, 79, 1089-1125

https://doi.org/10.1152/physrev.1999.79.4.1089

Article | Google Scholar

]

.

In this work, using single human platelets, we investigated the issues of the possible influence of the used fluorescent probe and the conditions of its entry into the cell on the observed calcium signal. As a result of this work, we conclude that the intensity of calcium mobilization in the platelet in response to activation correlates positively with the level of fluorescence of the fluorophore in the platelet.

Materials and methods

The following materials were obtained from the parenthesized sources: Human Thrombin (Hematologic Technologies, Essex Junction, Vermont, USA), Calbryte 590 AM, Fura Red AM, Fluo-4 AM, and DiOC6 (3) (Molecular probes, Eugene, State Oregon, USA), CellTracker Violet BMQC Dye (Invitrogen), S-Monovette Hirudin 1.6 ml hirudin tubes (SARSTEDT AG Co. KG, Germany), 4.5 ml citrate (3.8%) tubes (IMPROVACUTER, China). Potato apyrase, ADP and solution reagents were obtained from Sigma-Aldrich. VM64 antibodies were given as a gift to Prof. A.V. Mazurov (Federal State Budgetary Institution of National Medical Research Center of Cardiology, Moscow, RF). Materials for the manufacture of flow chambers were produced by GemaKor (Moscow, RF) and MedSil (Mytishchi, RF).

The whole blood of healthy volunteers (11 persons, 18-45 years of age) within 3 hours of the collection was used for all experiments. Blood was collected into 9 ml tubes containing 3,8% sodium citrate (1:9 v/v), or into 1.6 ml tubes containing R-hirudin (S-Monovette®, Sarstedt, Germany). Investigations were performed in accordance with the Declaration of Helsinki and approved by the CTP PCP RAS ethics committee (decision #1 from 12.01.2018), written informed consent was obtained from all donors.

9 different protocols (A-I) for loading calcium sensitive probes into platelets were used for this study. Protocols A-E are based on the sodium citrate-anticoagulated (3.8 % v/v) blood, while in protocols F-H hirudin (> 525 antithrombin units / ml) was used as the anticoagulant. 2 µM of Calbryte590-AM was used as a probe for protocols A, B, D, E, and H, protocol C used DIOC6, protocol F - Fluo4-AM (2 μM), protocol G - Fura Red- AM (2 μM), and protocol I – CellTracker Violet BMQC Dye (2 μM and 4 μM). Apyrase (0.3 U/ml) was used to prevent platelet pre-activation due to ADP, secreted by the red blood cells. Protocol D also contained Pluronic F-127 (0.04%) and in protocol E, 5 mM of MβCD (methyl-β-cyclodextrin) was added after 15 min of blood incubation with calcium probe. The resulting mixture was incubated for 30 minutes (A, B, D, E, F, G, H) or for 5 minutes (C) at 37°C (A, D, E, F, G, H) or at 25° C (B, C). Thus, protocol A can be used as a control for the CalBryte590 dye, protocol C for the DiOC6 dye, F for the Fluo4 dye, G for the Fura Red dye, and I – CellTracker Violet BMQC dye. Protocols A, B, D and E use different conditions for loading the CalBryte590 probe into platelets: B is characterized by a reduced temperature, D by the presence of Pluronic (a reagent to facilitate the entry of a hydrophobic AM probe into the cell), and Protocol E is characterized by the presence of MβCD (reagent for depletion platelet membrane with cholesterol).

Parallel-plate flow chambers, used here, were described earlier

[

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Clusterization of Inositol Trisphosphate Receptors Determines the Shape of the Calcium Oscillation Peak in Platelet Cytosol

F. Balabin, D. Morozova, A. Mayorov, A. Martyanov, M. Panteleev, A. Sveshnikova

Moscow University Physics Bulletin. 2018, 73, 526-533

https://doi.org/10.3103/S0027134918050041

Article | Google Scholar

]

. Monoclonal antibody to CD31 covered glass coverslips were used for platelet immobilization. Anti-CD31 antibody was isolated same as in

[

22

A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells

A. Mazurov, D. Vinogradov, N. Kabaeva, G. Antonova, Y. Romanov, T. VIasik, A. Antonov, V. Smirnov

Thrombosis and Haemostasis. 1991, 66, 494-499

https://doi.org/10.1055/s-0038-1646445

Article | Google Scholar

]

. Platelets were isolated from the whole blood upon perfusion through the flow chamber and interaction with the antibody covered glass. Unattached cells were washed away using tyrode’s calcium buffer and then platelet activation was monitored upon perfusion of the buffers, containing platelet activators. Nikon Eclipse Ti-E microscope with a CFI Apochromat TIRF 100XC Oil objective with 100-fold magnification and a numerical aperture of 1.49 was used in this study. Fluorescent probes were excited by laser at 405, 488 and 561 nm wavelength. Images were recorded using an Andor iXon3 EMCCD camera. The scheme of the experiment is given in Figure 1.

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.

Figure 1. 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.

ImageJ-Fiji was used for the analysis of the experimental data. Regions of interest (ROIs) of 2µm diameter were selected inside of the cells. At each timestep averaged fluorescence intensity was measured inside these ROIs. Based on this data, fluorescence intensity profiles were built (Fig. 2). Pre-activation of platelets has been assessed by identification of specific platelet types within the field of view (FOV) as has been previously described

[

23

Quantitative Assessment of Heterogeneity of Single Platelet Calcium Responses to Activation

Balabin FA.

Research and Practice in Thrombosis and Haemostasis. 2021, 5, PB1027

Article | Google Scholar

]

: (1) weak activation (less than five random single peaks per minute), (2) activation (multiple stochastic peaks, which usually do not form clusters ), (3) strong activation (clusters of peaks in the profile) and (4) a stably high response (changes in fluorescence intensity are mainly caused by bleaching or leakage of the fluorescent probe) (Fig. 2).

Observed fluorescence intensity profiles of individual platelets and their classification. (A) Group 1: the cell shows less than five random single peaks in 60 seconds of observation. The red line shows the baseline fluorescence intensity level; (B) Group 2: platelets are weakly activated, there are multiple stochastic peaks, which usually do not form clusters; (C) Group 3: platelet activation is intense, peaks are so frequent that individual peaks coalesce into clusters; (D) Group 4: Presumably, the cells show a persistently high concentration of calcium, any observed changes in fluorescence intensity are mainly caused by bleaching or leakage of the fluorescent dye; Orange boxes are clusters of peaks (clusters). Green shaded areas are persistent high calcium levels.

Figure 2. Observed fluorescence intensity profiles of individual platelets and their classification. (A) Group 1: the cell shows less than five random single peaks in 60 seconds of observation. The red line shows the baseline fluorescence intensity level; (B) Group 2: platelets are weakly activated, there are multiple stochastic peaks, which usually do not form clusters; (C) Group 3: platelet activation is intense, peaks are so frequent that individual peaks coalesce into clusters; (D) Group 4: Presumably, the cells show a persistently high concentration of calcium, any observed changes in fluorescence intensity are mainly caused by bleaching or leakage of the fluorescent dye; Orange boxes are clusters of peaks (clusters). Green shaded areas are persistent high calcium levels.

The amount of fluorophore, “loaded” into the platelet, was calculated as the average fluorescence of 10% of the points with the lowest fluorescence intensity (Fig. 3).

Figure 3. Example of selecting the 10% of the points with the lowest intensity in a fluorescence intensity profile.

Results and Discussion

Platelets are loaded heterogeneously by different fluorescent dyes

Different dyes load to platelets heterogeneously. The amount of dye in platelets of the same donor varies between the cells. To study this phenomenon, we determined the specificity of each probe. Platelets were immobilized on VM64 (anti-CD31). The amount of the probe in the platelet was assessed by the profile in the first 15 seconds of the experiment. Platelets from the same donor were loaded with four different fluorescent probes: CalBryte 590-AM (Protocol H), DiOC6(3) (Protocol C), Fura Red-AM (Protocol F) и Fluo-4-AM (Protocol G) (Fig. 4). For all dyes, there was a heterogeneity of the base platelet fluorescence intensity with a deviation of the fluorescence intensity from the median value by 2-6 times for 30% of the population. Interestingly, Calbryte 590-AM (Fig. 4А) и Fluo-4-AM (Fig. 4C) load more homogeneously than DiOC6 (Fig. 4B) and Fura Red-AM (Fig. 4D).

The observed phenomenon of specific loading of different probes may be related to the size and polarity of the fluorescent probe molecules. The presence of a difference in a potential of the platelets membrane

[

24,

Patch-clamp technique for studying ion channels in activated platelets

A. Kokhan, S. Zdanevich, I. Prokofev, I. Gorudko, E. Shamova

System Biology and Physiology Reports. 2021, 1, 3-11

https://doi.org/10.52455/sbpr.01.202101012

Article | Google Scholar

25

Calcium-activated potassium channels in human platelets.

M. Mahaut-Smith

The Journal of Physiology. 1995, 484, 15-24

https://doi.org/10.1113/jphysiol.1995.sp020644

Article | Google Scholar

]

facilitates the entry of cationic probes and hinders the anionic ones. However, heterogeneity of potential in a population can lead to heterogeneity of loading. Since DiOC6 (3) is a voltage-sensitive probe that does not contain the -AM part, its distribution in the platelet population precisely reflects this property of the platelet. (Fig. 4B). It has already been shown that Fluo-4 distributes heterogeneously in the platelets

[

26

A comparison of fluorescent Ca2+indicator properties and their use in measuring elementary and global Ca2+signals

D. Thomas, S. Tovey, T. Collins, M. Bootman, M. Berridge, P. Lipp

Cell Calcium. 2000, 28, 213-223

https://doi.org/10.1054/ceca.2000.0152

Article | Google Scholar

]

and it has been primarily associated with the compartmentalization of the dye. However, the causes for this heterogeneity are not fully understood. Perhaps, the compartmentalization of the dye may cause heterogeneity. Furthermore, it is known that low temperature of incubation and the usage of surfactants reduce the compartmentalization of the probes

[

27

Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart

K. Thompson, P. Dockery, R. Horobin

Biotechnic & Histochemistry. 2012, 87, 468-483

https://doi.org/10.3109/10520295.2012.703691

Article | Google Scholar

]

.

Baseline fluorescence intensity histograms for CalBryte 590 AM, DiOC6 (3), Fluo-4 AM and Fura Red. The fluorescence of the probes is normalized to the median. Each histogram is based on data analysis of n = 270 platelets from m = 3 healthy donors.

Figure 4. Baseline fluorescence intensity histograms for CalBryte 590 AM, DiOC6 (3), Fluo-4 AM and Fura Red. The fluorescence of the probes is normalized to the median. Each histogram is based on data analysis of n = 270 platelets from m = 3 healthy donors.

In order to find out the cause for the uneven loading of platelets with fluorescent probes, we used the CellTracker Violet BMQC dye (Fig. 5). Our purpose was to visualize the position of the cell in space. This substance is a derivative of coumarin, it can freely enter the cell and remain inside it for a long time because of binding with glutathione. We have no data that the fluorescence intensity of this dye depends on the concentration of calcium in the cytosol, hence it follows that the state of the plasma membrane plays the main role in the ingress of paint into the cell.

Figure 5. Histograms of baseline fluorescence intensity for the CellTracker Violet BMQC probe for 2 μM and 4 μM, normalized to median.

Platelet loading with a fluorescent probe depends on the experimental conditions

The loading of the fluorescent dye may be affected by the conditions of incubation. To test this, we compared the amount of probe in the platelet under different loading conditions: at room temperature (protocol B, see Methods), in the presence of a hydrophobic agent (protocol D) or when cholesterol is depleted from the membrane (protocol E) (Fig. 6). The statistical significance of differences in the number of "loaded" probe was calculated using the Mann-Whitney and Kolmogorov-Smirnov tests (Table 1).

All three samples do not belong to the same distribution law as the control one. Samples MβCD and 25 ° C for both tests give a p-value of less than 0.01, a sample with Pluronic F-127 for the Kolmogorov-Smirnov test gives a p-value of less than 0.01, and for the Mann-Whitney test gives a p-value of less than 0.05.

Dependence of the level of basic fluorescence intensity on the conditions of the experiment. Green arrow - mean value, orange bar - median value, “box” - from 25% to 75% of the population, “wiskers” - 95% confidence interval. МβCD increases the fluorescence intensity of the Calbryte 590 AM probe compared to the control. A decrease in the activation temperature from 37 ° C to 25 ° C slows down the loading of cells with the probe. Pluronic F-127 forces the probe to spread more evenly throughout the cells than under control conditions, but does not affect the platelet membrane itself. Data are given for n = 360 platelets from m = 10 healthy donors.

Figure 6. Dependence of the level of basic fluorescence intensity on the conditions of the experiment. Green arrow - mean value, orange bar - median value, “box” - from 25% to 75% of the population, “wiskers” - 95% confidence interval. МβCD increases the fluorescence intensity of the Calbryte 590 AM probe compared to the control. A decrease in the activation temperature from 37 ° C to 25 ° C slows down the loading of cells with the probe. Pluronic F-127 forces the probe to spread more evenly throughout the cells than under control conditions, but does not affect the platelet membrane itself. Data are given for n = 360 platelets from m = 10 healthy donors.

Table 1. Comparison of the parameters of the control group with the rest.

Incubation at room temperature, compared with incubation at 37 ° C, reduces the average and median fluorescence intensities of cells by more than 100%. This result is consistent with the data that the permeability of the cell membrane is dependent on temperature

[

28

Using quin2 in cell suspensions

T. Rink, T. Pozzan

Cell Calcium. 1985, 6, 133-144

https://doi.org/10.1016/0143-4160(85)90040-5

Article | Google Scholar

]

.

Pluronic F-127 reduces the average amount of the probe in the platelet (by 240%) and the median (by 23%). The upper limit of the 95% confidence interval is decreased by more than 550%. Pluronic is a surfactant that increases the solubility of probes in the AM form

[

29

Adsorption of Pluronic F-127 on Surfaces with Different Hydrophobicities Probed by Quartz Crystal Microbalance with Dissipation

M. Nejadnik, A. Olsson, P. Sharma, H. van der Mei, W. Norde, H. Busscher

Langmuir. 2009, 25, 6245-6249

https://doi.org/10.1021/la9001169

Article | Google Scholar

]

. Thus, the distribution of the probe in the cell becomes more uniform, and the number of overloaded platelets decreases (mean value decreases, the median value does not change).

Incubation with МβCD increases the mean (by 17.7%) and median (by 72.9%) values of the fluorescence intensity in comparison with the control sample. MβCD increases platelet membrane fluidity by decreasing cholesterol concentration

[

30

Effect of cholesterol on the structure of a phospholipid bilayer

F. de Meyer, B. Smit

Proceedings of the National Academy of Sciences. 2009, 106, 3654-3658

https://doi.org/10.1073/pnas.0809959106

Article | Google Scholar

]

, while not leading to cell activation

[

31,

The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses

A. Moscardó, J. Vallés, A. Latorre, M. Santos

FEBS Letters. 2014, 588, 3154-3159

https://doi.org/10.1016/j.febslet.2014.06.057

Article | Google Scholar

32

Role of in vitro cholesterol depletion in mediating human platelet aggregation

S. Grgurevich, R. Krishnan, M. White, L. Jennings

Journal of Thrombosis and Haemostasis. 2003, 1, 576-586

https://doi.org/10.1046/j.1538-7836.2003.00087.x

Article | Google Scholar

]

. The observed values of the amount of the probe show that MβCD promotes the dye to pass into the cell. That leads to a suggestion that the amount of the probe depends on the state of the membrane.

MβCD shortens the time of loading cells with a fluorescent probe

Cholesterol of the membrane affects the loading of the platelets with a probe. To investigate this, the platelet loading rate was monitored with a Calbryte 590-AM fluorescent probe without MβCD (Protocol A) and in presence of MβCD (Protocol E) (Fig. 7). MβCD increases the rate of entry of the probe into the cell by approximately 2 times. And the increase in fluorescence intensity in the cytosol begins 2 times faster than under control conditions.

Comparison of median fluorescence intensity versus time in the presence and absence of MβCD. Orange - with MβCD, blue – without MβCD. Light orange and light blue are the interquartile ranges respectively. Data are presented for n = 90 platelets from m = 3 healthy donors.

Figure 7. Comparison of median fluorescence intensity versus time in the presence and absence of MβCD. Orange - with MβCD, blue – without MβCD. Light orange and light blue are the interquartile ranges respectively. Data are presented for n = 90 platelets from m = 3 healthy donors.

The amount of the probe in the platelet correlates with its response to stimulation

The fluorescent probe does not evenly enter platelets in the population regardless of the dye and loading conditions. But it remains unclear whether this phenomenon is for the study of calcium signaling in platelets. To investigate this, we checked if the baseline fluorescence intensity of the probe and activation of platelet are related (Fig. 8). The amount of the probe in the platelet was assessed according to the profile in the first 15 seconds of the experiment. The division of platelets into groups by activation was proceeded according to the profiles taken within 5 minutes. Figure 8 shows the obtained dependence of the activation group on the amount of probe (the “lower limit” of the fluorescence intensity of CalBryte590 in the cell). It turned out that platelets from the fourth activation group had, on average, 10 times more probe than platelets from the third group. This can be explained by the possible activation of platelets of the fourth group at the time of the beginning of observation. However, platelets from the third activation group also had 7 times more probe than platelets from the second group. The second group had 1.5 times more probe than platelets from the first group. Since platelets of groups 1-3 have a pronounced baseline level (Fig. 2), we conclude that the degree of platelet activation correlates with the amount of fluorophore.

Figure 8. Dependence of the cell response group number on the intensity of its base fluorescence. Green arrow - mean value, orange line - median value, "box" - from 25% to 75% of the population, "whiskers" - 95% confidence interval. In total, n = 122 platelets from m = 9 healthy donors were analyzed.

To clarify the observed phenomenon, we decided to estimate the probability of obtaining a platelet with a given value of baseline fluorescence in each of the activation groups using the available sample (Fig. 9). A platelet with a high baseline fluorescence intensity is more likely to undergo "strong" activation than a platelet with a low baseline fluorescence intensity.

Thus, it can be argued that evaluation of the response of platelets by measuring calcium concentration (by means of flow cytometry or spectrofluorimetry) is shifted in suspension. This shift occurs because the platelets, that are strongly activated, come to the fore. Thus, the overall response of the suspension appears to be stronger than under physiological conditions.

Figure 9. The probability of a platelet getting into the activation group, depending on the "lower limit" of the fluorescence intensity. The probability is calculated on the basis of data on n = 122 platelets from m = 9 healthy donors.

Conclusion

We have shown that platelets load heterogeneously with CalBryte 590 AM. This heterogeneity is not accidental. It was found that the probability of experiencing "strong" activation for a platelet with a high baseline fluorescence intensity is higher than for a platelet with a low baseline fluorescence intensity. The heterogeneity of platelet loading with a probe, as well as the loading process itself, can be influenced by various agents and experimental conditions. We observed this with the CalBryte 590 AM fluorescent dye. Incubation of the cells with the probe on 25°C instead of on 37°C slows down the loading. Pluronic F-127 makes the probe distribute more homogeneously throughout the cells and it does not affect the platelet membrane. MβCD increases the fluorescence intensity of the probe in the cytosol compared to control. It also increases the rate of platelet loading by the probe.

Heterogeneous cell loading is not unique to the CalBryte 590 AM probe. We have shown that fluorescent probes DiOC6 (3), Fura Red AM, Fluo-4 AM also load heterogeneously into platelets. The heterogeneity of DiOC6 loading is comparable to that of other probes

[

3

Platelet Integrin αIIbβ3: Mechanisms of Activation and Clustering; Involvement into the Formation of the Thrombus Heterogeneous Structure

V. Kaneva, A. Martyanov, D. Morozova, M. Panteleev, A. Sveshnikova

Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology. 2019, 13, 97-110

https://doi.org/10.1134/S1990747819010033

Article | Google Scholar

]

and it indicates that there is a noticeable heterogeneity of the potential difference on platelet membranes. It is assumed that this partially explains the phenomenon of heterogeneity of cell loading in general.

However, our data are insufficient to determine the cause for the heterogeneity of platelet loading to occur. A further investigation is required to study the combinations of the effects of the parameters that we used. Furthermore, size of the probe molecule, its quantum-chemical properties are of high interest to be investigated. However, it is possible to identify a number of factors that significantly affect the amount of a fluorescent probe in a living cell. First of all, the composition of the plasma membrane is important. We showed that hydrophobic low molecular weight substances pass through the membrane when it contains more unsaturated fatty acids. The amount of the loaded dye depends on the temperature of incubation. Secondly, the physicochemical state of the probe outside the cell has an effect. Moreover, the activity of nonspecific esterases, which cut off the AM-“tails”, is important. There are also two more factors that are sensitive to cell activation: the presence of nonspecific channels or carriers for both the AM-form of the probe and the working form, and the sensitivity of one of the probe forms to the potential difference on the membrane as a result of its charge.

Funding

The study was supported by RFBR and the Royal Society of London (RS) grant, project number 21-51-10005

References of this article:

Hemostasis and thrombosis beyond biochemistry: roles of geometry, flow and diffusion
M. Panteleev, N. Dashkevich, F. Ataullakhanov
Thrombosis Research. 2015, 136, 699-711
https://doi.org/10.1016/j.thromres.2015.07.025
Article|Google Scholar
Platelets and hemostasis
Panteleev M.A., Sveshnikova A.N.
Oncohematology. 2014, 9(2), 65-73
https://doi.org/10.17650/1818-8346-2014-9-2-65-73
Article|Google Scholar
Platelet Integrin αIIbβ3: Mechanisms of Activation and Clustering; Involvement into the Formation of the Thrombus Heterogeneous Structure
V. Kaneva, A. Martyanov, D. Morozova, M. Panteleev, A. Sveshnikova
Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology. 2019, 13, 97-110
https://doi.org/10.1134/S1990747819010033
Article|Google Scholar
Platelet functional responses and signalling: the molecular relationship. Part 1: responses.
A. Sveshnikova, M. Stepanyan, M. Panteleev
Systems Biology and Physiology Reports. 2021, 1, 20-28
https://doi.org/10.52455/sbpr.01.202101014
Article|Google Scholar
Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation
N. Topalov, Y. Kotova, S. Vasil’ev, M. Panteleev
British Journal of Haematology. 2012, 157, 105-115
https://doi.org/10.1111/j.1365-2141.2011.09021.x
Article|Google Scholar
Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting
N. Podoplelova, A. Sveshnikova, Y. Kotova, A. Eckly, N. Receveur, D. Nechipurenko, S. Obydennyi, I. Kireev, C. Gachet, F. Ataullakhanov, P. Mangin, M. Panteleev
Blood. 2016, 128, 1745-1755
https://doi.org/10.1182/blood-2016-02-696898
Article|Google Scholar
Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface
D. Nechipurenko, N. Receveur, A. Yakimenko, T. Shepelyuk, A. Yakusheva, R. Kerimov, S. Obydennyy, A. Eckly, C. Léon, C. Gachet, E. Grishchuk, F. Ataullakhanov, P. Mangin, M. Panteleev
Arteriosclerosis, Thrombosis, and Vascular Biology. 2019, 39, 37-47
https://doi.org/10.1161/ATVBAHA.118.311390
Article|Google Scholar
PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets
M. HARPER, A. POOLE
Journal of Thrombosis and Haemostasis. 2011, 9, 1599-1607
https://doi.org/10.1111/j.1538-7836.2011.04393.x
Article|Google Scholar
Heterogeneity of Integrin αIIbβ3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis
A. Martyanov, D. Morozova, M. Sorokina, A. Filkova, D. Fedorova, S. Uzueva, E. Suntsova, G. Novichkova, P. Zharkov, M. Panteleev, A. Sveshnikova
International Journal of Molecular Sciences. 2020, 21, 3035
https://doi.org/10.3390/ijms21093035
Article|Google Scholar
Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N
S. SAGE, N. PUGH, M. MASON, A. HARPER
Journal of Thrombosis and Haemostasis. 2011, 9, 540-551
https://doi.org/10.1111/j.1538-7836.2010.04159.x
Article|Google Scholar
Platelet functional responses and signalling: the molecular relationship. Part 2: receptors.
A. Martyanov, M. Panteleev
Systems Biology and Physiology Reports. 2021, 1, 13-30
https://doi.org/10.52455/sbpr.01.202103013
Article|Google Scholar
Why Calcium? How Calcium Became the Best Communicator
E. Carafoli, J. Krebs
Journal of Biological Chemistry. 2016, 291, 20849-20857
https://doi.org/10.1074/jbc.R116.735894
Article|Google Scholar
The calcium-signalling saga: tap water and protein crystals
E. Carafoli
Nature Reviews Molecular Cell Biology. 2003, 4, 326-332
https://doi.org/10.1038/nrm1073
Article|Google Scholar
Transient Receptor Potential Channels Function as a Coincidence Signal Detector Mediating Phosphatidylserine Exposure
M. Harper, J. Londoño, K. Quick, J. Londoño, V. Flockerzi, S. Philipp, L. Birnbaumer, M. Freichel, A. Poole
Science Signaling. 2013, 6,
https://doi.org/10.1126/scisignal.2003701
Article|Google Scholar
Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling
A. Sveshnikova, A. Balatskiy, A. Demianova, T. Shepelyuk, S. Shakhidzhanov, M. Balatskaya, A. Pichugin, F. Ataullakhanov, M. Panteleev
Journal of Thrombosis and Haemostasis. 2016, 14, 2045-2057
https://doi.org/10.1111/jth.13442
Article|Google Scholar
Roles of phospholipase C and Ca(2+)-ATPase in calcium responses of single, fibrinogen-bound platelets.
J. Heemskerk, P. Vis, M. Feijge, J. Hoyland, W. Mason, S. Sage
Journal of Biological Chemistry. 1993, 268, 356-363
https://doi.org/10.1016/S0021-9258(18)54158-2
Article|Google Scholar
Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets
J. Heemskerk, J. Hoyland, W. Mason, S. Sage
Biochemical Journal. 1992, 283, 379-383
https://doi.org/10.1042/bj2830379
Article|Google Scholar
Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation
S. Obydennyy, A. Sveshnikova, F. Ataullakhanov, M. Panteleev
Journal of Thrombosis and Haemostasis. 2016, 14, 1867-1881
https://doi.org/10.1111/jth.13395
Article|Google Scholar
Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging
T. Ha, P. Tinnefeld
Annual Review of Physical Chemistry. 2012, 63, 595-617
https://doi.org/10.1146/annurev-physchem-032210-103340
Article|Google Scholar
Measurement of Intracellular Calcium
A. Takahashi, P. Camacho, J. Lechleiter, B. Herman
Physiological Reviews. 1999, 79, 1089-1125
https://doi.org/10.1152/physrev.1999.79.4.1089
Article|Google Scholar
Clusterization of Inositol Trisphosphate Receptors Determines the Shape of the Calcium Oscillation Peak in Platelet Cytosol
F. Balabin, D. Morozova, A. Mayorov, A. Martyanov, M. Panteleev, A. Sveshnikova
Moscow University Physics Bulletin. 2018, 73, 526-533
https://doi.org/10.3103/S0027134918050041
Article|Google Scholar
A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells
A. Mazurov, D. Vinogradov, N. Kabaeva, G. Antonova, Y. Romanov, T. VIasik, A. Antonov, V. Smirnov
Thrombosis and Haemostasis. 1991, 66, 494-499
https://doi.org/10.1055/s-0038-1646445
Article|Google Scholar
Quantitative Assessment of Heterogeneity of Single Platelet Calcium Responses to Activation
Balabin FA.
Research and Practice in Thrombosis and Haemostasis. 2021, 5, PB1027
Google Scholar
Patch-clamp technique for studying ion channels in activated platelets
A. Kokhan, S. Zdanevich, I. Prokofev, I. Gorudko, E. Shamova
System Biology and Physiology Reports. 2021, 1, 3-11
https://doi.org/10.52455/sbpr.01.202101012
Article|Google Scholar
Calcium-activated potassium channels in human platelets.
M. Mahaut-Smith
The Journal of Physiology. 1995, 484, 15-24
https://doi.org/10.1113/jphysiol.1995.sp020644
Article|Google Scholar
A comparison of fluorescent Ca2+indicator properties and their use in measuring elementary and global Ca2+signals
D. Thomas, S. Tovey, T. Collins, M. Bootman, M. Berridge, P. Lipp
Cell Calcium. 2000, 28, 213-223
https://doi.org/10.1054/ceca.2000.0152
Article|Google Scholar
Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart
K. Thompson, P. Dockery, R. Horobin
Biotechnic & Histochemistry. 2012, 87, 468-483
https://doi.org/10.3109/10520295.2012.703691
Article|Google Scholar
Using quin2 in cell suspensions
T. Rink, T. Pozzan
Cell Calcium. 1985, 6, 133-144
https://doi.org/10.1016/0143-4160(85)90040-5
Article|Google Scholar
Adsorption of Pluronic F-127 on Surfaces with Different Hydrophobicities Probed by Quartz Crystal Microbalance with Dissipation
M. Nejadnik, A. Olsson, P. Sharma, H. van der Mei, W. Norde, H. Busscher
Langmuir. 2009, 25, 6245-6249
https://doi.org/10.1021/la9001169
Article|Google Scholar
Effect of cholesterol on the structure of a phospholipid bilayer
F. de Meyer, B. Smit
Proceedings of the National Academy of Sciences. 2009, 106, 3654-3658
https://doi.org/10.1073/pnas.0809959106
Article|Google Scholar
The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses
A. Moscardó, J. Vallés, A. Latorre, M. Santos
FEBS Letters. 2014, 588, 3154-3159
https://doi.org/10.1016/j.febslet.2014.06.057
Article|Google Scholar
Role of in vitro cholesterol depletion in mediating human platelet aggregation
S. Grgurevich, R. Krishnan, M. White, L. Jennings
Journal of Thrombosis and Haemostasis. 2003, 1, 576-586
https://doi.org/10.1046/j.1538-7836.2003.00087.x
Article|Google Scholar