Patch-clamp technique for studying ion channels in activated platelets

Mammalian platelets are small anucleate cells that play a major role in thrombosis and hemostasis. In the past couple of decades, it has been identified that both ion channels and membrane potential play a pivotal role in platelet physiological and pathophysiological functions. Before the introduction of patch-clamp methodology, researchers used fluorescence potential sensitive dyes

[

1,

Dye indicators of membrane potential

Waggoner, A. S.

Annual review of biophysics and bioengineering. 1979, 8(1), 47-68

https://doi.org/10.1146/annurev.bb.08.060179.000403

Article | Google Scholar

2,

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes

Felix, J. P., Williams, B. S., Priest, B. T., Brochu, R. M., Dick, I. E., Warren, V. A., ... & Garcia, M. L.

Assay and drug development technologies. 2004, 2(3), 260-268

https://doi.org/10.1089/1540658041410696

Article | Google Scholar

3

Nitrate-selective optical sensor applying a lipophilic fluorescent potential-sensitive dye

Huber, C., Klimant, I., Krause, C., Werner, T., & Wolfbeis, O. S.

Analytica chimica acta. 2001, 449(1-2), 81-93

https://doi.org/10.1016/S0003-2670(01)01363-0

Article | Google Scholar

]

and radioactive isotopes

[

4,

A Ca-dependent K channel in “luminal” membranes from the renal outer medulla

Burnham, C., Braw, R., & Karlish, S. J. D.

The Journal of membrane biology. 1986, 93(2), 177-186

https://doi.org/10.1007/BF01870809

Article | Google Scholar

5,

A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles

Garty, H., Rudy, B., & Karlish, S. J.

Journal of Biological Chemistry. 1983, 258(21), 13094-13099

https://doi.org/10.1016/S0021-9258(17)44085-3

Article | Google Scholar

6,

Amiloride blockable sodium fluxes in toad bladder membrane vesicles

Garty, H.

The Journal of membrane biology. 1984, 82(3), 269-279

https://doi.org/10.1007/BF01871636

Article | Google Scholar

7

Identification and reconstitution of a Na+/K+/Cl− cotransporter and K+ channel from luminal membranes of renal red outer medulla

Burnham, C., Karlish, S. J. D., & Jørgensen, P. L.

Biochimica et Biophysica Acta (BBA)-Biomembranes. 1985, 821(3), 461-469

https://doi.org/10.1016/0005-2736(85)90051-3

Article | Google Scholar

]

to study ion conductance and changes in membrane potential. The introduction of the patch-clamp technique

[

8,

The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes

Neher, E., Sakmann, B., & Steinbach, J. H.

Pflügers Archiv. 1978, 375(2), 219-228

https://doi.org/10.1007/BF00584247

Article | Google Scholar

9

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., & Sigworth, F. J.

Pflügers Archiv. 1981, 391(2), 85-100

https://doi.org/10.1007/BF00656997

Article | Google Scholar

]

provided tools for direct assessment of the conductance of single ion channels or the whole membrane, as well as the way to precisely measure the value of the membrane potential even of such small cells as platelets.

Patch-clamp is an electrophysiological technique, which allows measurement of ion currents through single channels by clamping voltage of an isolated piece of the cell membrane (or whole-cell). There are several configurations in this technique. Getting a giga-seal between the pipette and the cell membrane leads to cell-attached configuration, allowing the measurement of ion currents through single channels under the patch pipette. Rupture of cell membrane under patch pipette leads to whole-cell configuration, in which both average currents across the entire surface area of the cell and kinetics of membrane potential can be measured. Other widely used patch configurations are the inside-out and outside-out configurations, in which either the intracellular or extracellular surface of the membrane patch is exposed to the extracellular solution.

The application of the patch-clamp technique to platelets shed light on various mechanisms of their functional activity. For example, it was shown that the most abundant ion channel in the platelet membrane is voltage-dependent potassium channel K_v 1.3, and its pivotal role in maintaining resting membrane potential was demonstrated

[

10,

A patch‐clamp study of mammalian platelets and their voltage‐gated potassium current.

Maruyama, Y.

The Journal of physiology. 1987, None, None

https://doi.org/10.1113/jphysiol.1987.sp016750

Article | Google Scholar

11

Voltage‐gated potassium channels and the control of membrane potential in human platelets

Mahaut‐Smith, M. P., Rink, T. J., Collins, S. C., & Sage, S. O.

The Journal of physiology. 1990, 428(1), 723-735

https://doi.org/10.1113/jphysiol.1990.sp018237

Article | Google Scholar

]

. Patch-clamp helped to identify calcium-dependent potassium channels K_Ca 3.1 and to reveal their role in maintaining the driving force for Ca²⁺ during platelet activation

[

12

Calcium‐activated potassium channels in human platelets.

Mahaut-Smith, M. P.

The Journal of physiology. 1995, 484(1), 15-24

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

Article | Google Scholar

]

. Furthermore, several important modulators of calcium signaling were revealed in platelets and megakaryocytes using this technique: the receptor-operated cationic P2X channels

[

13,

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique

Mahaut-Smith, M. P., Sage, S. O., & Rink, T. J.

Journal of Biological Chemistry. 1992, 267(5), 3060-3065

https://doi.org/10.1016/S0021-9258(19)50694-9

Article | Google Scholar

14

Receptor-activated single channels in intact human platelets

Mahaut-Smith, M. P., Sage, S. O., & Rink, T. J.

Journal of Biological Chemistry. 1990, 265(18), 10479-10483

https://doi.org/10.1016/S0021-9258(18)86972-1

Article | Google Scholar

]

as well as CRAC-channels Orai-1

[

15,

Three cation influx currents activated by purinergic receptor stimulation in rat megakaryocytes

Somasundaram, B., & Mahaut-Smith, M. P.

The Journal of physiology. 1994, 480(2), 225-231

https://doi.org/10.1113/jphysiol.1994.sp020355

Article | Google Scholar

16,

Primaquine, an inhibitor of vesicular transport, blocks the calcium-release-activated current in rat megakaryocytes

Somasundaram, B., Norman, J. C., & Mahaut-Smith, M. P.

Biochemical Journal. 1995, 309(3), 725-729

https://doi.org/10.1042/bj3090725

Article | Google Scholar

17

Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes

Tolhurst, G., Carter, R. N., Amisten, S., Holdich, J. P., Erlinge, D., & Mahaut-Smith, M. P.

Platelets. 2008, 19(4), 308-313

https://doi.org/10.1080/09537100801935710

Article | Google Scholar

]

. One of the essential applications of the patch-clamp technique is the measurement of membrane potential. Previous studies, using fluorescent techniques, showed that platelet activation by various agonists leads to different changes in membrane potential. Thus, platelet activation by thrombin (more than 0.1 U/ml) leads to depolarization of the platelet membrane, while ADP (0.3-30 µM) induces hyperpolarization followed by depolarization

[

18

Probes of transmembrane potentials in platelets: changes in cyanine dye fluorescence in response to aggregation stimuli.

Home, W. C., & Simons, E. R.

Blood. 1978, 51(4), 741-749

https://doi.org/10.1182/blood.V51.4.741.741

Article | Google Scholar

]

. More recent experiments using patch-clamp showed that membrane potential of megakaryocytes and possibly platelets can oscillate during activation with ADP

[

19,

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

Mason, M. J., Simpson, A. K., Mahaut-Smith, M. P., & Robinson, H. P. C.

Biophysical journal. 2005, 88(1), 739-750

https://doi.org/10.1529/biophysj.104.049866

Article | Google Scholar

20

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes

Mason, M. J., Hussain, J. F., & Mahaut-Smith, M. P.

The Journal of Physiology. 2000, 524(Pt 2), 437

https://doi.org/10.1111/j.1469-7793.2000.00437.x

Article | Google Scholar

]

. Furthermore, changes of the membrane potential of megakaryocytes can directly modulate intracellular calcium spiking

[

20

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes

Mason, M. J., Hussain, J. F., & Mahaut-Smith, M. P.

The Journal of Physiology. 2000, 524(Pt 2), 437

https://doi.org/10.1111/j.1469-7793.2000.00437.x

Article | Google Scholar

]

. There is also some evidence of direct voltage control of G-protein coupled receptors

[

21,

The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling

Rinne, A., Mobarec, J. C., Mahaut-Smith, M., Kolb, P., & Bünemann, M.

Science Signaling. 2015, 8(401), ra110-ra110

https://doi.org/10.1126/scisignal.aac7419

Article | Google Scholar

22

Direct voltage control of signaling via P2Y1 and other Gαq-coupled receptors

Martinez-Pinna, J., Gurung, I. S., Vial, C., Leon, C., Gachet, C., Evans, R. J., & Mahaut-Smith, M. P.

Journal of Biological Chemistry. 2005, 280(2), 1490-1498

https://doi.org/10.1074/jbc.M407783200

Article | Google Scholar

]

.

Transcriptomic analysis

[

23

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Wright, J. R., Amisten, S., Goodall, A. H., & Mahaut-Smith, M. P.

Thrombosis and haemostasis. 2016, 116(2), 272

https://doi.org/10.1160/th15-11-0891

Article | Google Scholar

]

as well as recent studies of platelets and megakaryocytes

[

24

Expression and functional characterization of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 in megakaryocytes and platelets

Balduini, A., Fava, C., Di Buduo, C. A., Abbonante, V., Meneguzzi, A., Soprano, P. M., ... & Minuz, P.

J Thromb Haemost. 2021, None, None

https://doi.org/10.1111/jth.15269

Article | Google Scholar

]

evidence for the existence of ion channels unidentified earlier in platelets. This suggests that the application of the patch-clamp methodology to platelets is still of great importance for understanding the mechanisms underlying the variety of functions of these minute cells.

However, no matter how helpful this method is, there are many challenges concerning applying it to platelets. These cells are very tiny and fragile, which makes some of the developed patch-clamp techniques unusable. For example, there is no documented evidence that outside-out configuration is possible for platelets; furthermore, there is an opinion that there is no need for it as whole-cell configuration for platelets is essentially outside-out

[

25

Patch-clamp recordings of electrophysiological events in the platelet and megakaryocyte.

Mahaut-Smith, M. P.

In Platelets and Megakaryocytes . 2004, None, 277-299

https://doi.org/10.1385/1-59259-783-1:277

Article | Google Scholar

]

. It is also worth saying, that obtaining whole-cell configuration for platelets is sometimes pretty challenging, and oftentimes leads to the deterioration of the patch. Evidence exists that adding ATP to the pipette solution in the absence of calcium ions helps to obtain this configuration

[

10

A patch‐clamp study of mammalian platelets and their voltage‐gated potassium current.

Maruyama, Y.

The Journal of physiology. 1987, None, None

https://doi.org/10.1113/jphysiol.1987.sp016750

Article | Google Scholar

]

. A more prominent way to achieve whole-cell is to use nystatin (perforated patch-clamp technique)

[

13

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique

Mahaut-Smith, M. P., Sage, S. O., & Rink, T. J.

Journal of Biological Chemistry. 1992, 267(5), 3060-3065

https://doi.org/10.1016/S0021-9258(19)50694-9

Article | Google Scholar

]

, but it also has several limitations.

Difficulties in working with platelets, their fragileness, and small size led researchers to the use of megakaryocytes and corresponding cell lines as model cells in patch-clamp studies instead of platelets

[

26,

Interplay between P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling

Tolhurst, G., Vial, C., Léon, C., Gachet, C., Evans, R. J., & Mahaut-Smith, M. P.

Blood. 2005, 106(5), 1644-1651

https://doi.org/10.1182/blood-2005-02-0725

Article | Google Scholar

27

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte

Carter, R. N., Tolhurst, G., Walmsley, G., Vizuete‐Forster, M., Miller, N., & Mahaut‐Smith, M. P.

The Journal of physiology. 2006, 576(1), 151-162

https://doi.org/10.1113/jphysiol.2006.113886

Article | Google Scholar

]

. From one side it helps to achieve results that previously were very hard to obtain with platelets. On the other side, besides the similarities of megakaryocytes and platelets in regards to the receptor profiling, main signaling pathways, and ion channels, one can’t be sure, that obtained results on megakaryocytes can be directly transferred to platelets.

That is why this study aims to assess the difficulties of application of the patch-clamp technique to platelets, and suggest possible solutions, as well as to demonstrate the possibilities of the proposed method in regards to studying the single-channel currents during platelet activation.

Materials and methods

Chemicals

Sodium citrate, HEPES, ADP, glucose, ionomycin, and apyrase were purchased from Sigma-Aldrich (St. Louis, USA). Prostacycline was obtained from Santa Cruz (Dallas, USA).

Solutions

NaCl-based extracellular buffer saline (external BS) contained: 150 mM NaCl, 10 mM KCl, 10 mM HEPES, 10 mM glucose, titrated to pH 7.35 with NaOH. KCl-based pipette buffer saline (pipette BS) contained: 150 mM KCl, 10 mM NaCl, 10 mM HEPES, 2 mM CaCl₂, 2 mM MgCl₂, titrated to pH 7.2 with KOH. In the ion exchange experiments, KCl was replaced by an equimolar amount of NaCl in pipette BS. The osmolarity of solutions was brought to 300 mOsm for extracellular saline and 270 for pipette saline with the MilliQ water. All solutions were filtered through 0.22 μm syringe filters.

Cell preparation

Venous blood of healthy volunteers was collected into 10 mL tubes with 3.8 % (w/v) Na-citrate at 9 to 1 ratio, 0.5 μM PGI₂, 0.3 U/mL of apyrase and centrifuged at 100g for 7 minutes. 250 μL of platelet-rich plasma was mixed with 1.25 mL of extracellular saline, 0.5 mL of Na-citrate (pH 5.5), 0.5 μM PGI₂, 0.3 U/mL apyrase, and then centrifuged at 200 g for 5 min. Cells were resuspended in NaCl-based external BS. All manipulations were performed in compliance with the Declaration of Helsinki.

Patch pipettes

Patch pipettes were pulled from filamented borosilicate glass capillaries (0.86 mm ID, 1.5 mm OD). (HEKA Instruments) on a Sutter Instruments Brown P-97 pipette puller, and then heat polished on the same puller as described by the manufacturer. The resistance of patch pipettes filled with KCl-based pipette BS was 7-15 MΩ.

Electrophysiological recordings

Cell-attached and inside-out patch-clamp recordings were carried out in voltage-clamp and current-clamp modes using HEKA EPS 8 amplifier (HEKA Elektronic GmbH). Because all experiments were aimed at studying single-channel events, no series resistance compensation or correction for liquid junction potential were made. The experimental chamber was earthed directly through Ag/AgCl wire placed aside of the cells. The pipette was handled with Sutter MP-225 motorized micromanipulator. The plastic chamber was placed on the stage of the upright microscope (Olympus IX51WI, Tokyo, Japan) with an overall magnification of 400x. Data was filtered at 0.7 kHz using a built-in low-pass Bessel filter and digitized directly to the personal computer through B-381 AC/DC converter. Data was recorded at 1 kHz using a custom acquisition program on Matlab 2004 (The MathWorks, Inc.). Obtained data was analyzed using Origin 8.1 (Origin Lab, Northampton, Massachusetts, USA), WinEDR v. 3.9.1 (University of Strathclyde), and Spectragryph Software (F. Menges "Spekwin32 - optical spectroscopy software", Version 1.2.14, 2020, http://www.effemm2.de/spekwin/).

Activation of the cells

To avoid early platelet activation during platelet contact with the glass capillary (patch pipette), the process of gigaseal formation was performed in NaCl-based external BS in the absence of Ca²⁺. After that, 100 μL of extracellular suspension was mixed with CaCl₂ and agonist, and the experimental chamber was perfused with that mix (final CaCl₂concentration in bath solution was 2 mM unless otherwise specified).

Results and discussion

Patch pipettes and electronic noise

Usually, for small size cells it is recommended to use thick-walled capillaries for patch pipette production (for example, 0.86 mm ID, 1.5 mm OD)

[

28

Patch clamp techniques for single channel and whole-cell recording

Ogden, D., & Stanfield, P.

In Microelectrode techniques: the Plymouth workshop handbook . 1994, None, 53-78

Article | Google Scholar

]

. Theoretically, thin-walled pipettes also can be used, but the process of pipette production with a small aperture suitable for platelets is much harder. However, when using thick-walled pipettes, there are several obstacles, the main of which is the increased level of electronic noise that comes from the dielectric properties of glass

[

]

. Since many of the single-channel currents have amplitudes in the range of 0.5 to 2 pA, increased noise levels can obscure these events. In some patch-clamp studies, the reduction of noise level was achieved by covering the pipette tip with special silicon elastomer Sylgard which reduced pipette capacitance

[

9

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., & Sigworth, F. J.

Pflügers Archiv. 1981, 391(2), 85-100

https://doi.org/10.1007/BF00656997

Article | Google Scholar

]

. However, in our experiments, the necessary and sufficient requirements for single ion channel recordings with the amplitudes down to 0.4-0.5 pA, and in some cases even lower were: the proper grounding of the equipment, including Faraday cage, the seal resistance higher than 20 GΩ and gain level of the amplifier of 100 mV/pA or higher.

Obtaining gigaohm seal

2 μL of platelet suspension was placed on the bottom of the plastic chamber made out of 12-well cell culture plates with 700 μL of NaCl-based external BS, containing 1 mM of MgCl₂ (in the absence of Ca²⁺). Platelets were allowed to settle to the bottom of the chamber. Patch pipette was moved up to 1-2 mm of the chamber bottom with a motorized micromanipulator. Then 40x water-immersion objective of the upright microscope was moved into the suspension, and the tip of the pipette appeared on the screen. Only floating platelets were patched.

The success rate of the patch relies on several factors. Firstly, the pipette tip should always be clean, thus we made pipettes only before the experiment. And secondly, extracellular and pipette solutions should always be properly filtrated. Other important factors for successful gigaseal formation are the presence of the divalent cation (namely calcium and magnesium) in the pipette solution, and the difference in osmolarity between the pipette and extracellular saline’s at around 10% (30 mOsm), as was suggested by Neher and Sakmann in their pioneer work

[

9

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., & Sigworth, F. J.

Pflügers Archiv. 1981, 391(2), 85-100

https://doi.org/10.1007/BF00656997

Article | Google Scholar

]

. Previously it was shown both theoretically and with the help of model membranes, that divalent cations greatly enhance the contact between the cell membrane and capillary glass

[

29

Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording

Priel, A., Gil, Z., Moy, V. T., Magleby, K. L., & Silberberg, S. D.

Biophysical journal. 2007, 92(11), 3893-3900

https://doi.org/10.1529/biophysj.106.099119

Article | Google Scholar

]

. Another important factor is the level of pH in the pipette solution, because H⁺, as well as Ca²⁺ and Mg²⁺, also greatly improves the gigaohm seal formation by enhancing the strength of adhesion between cell membrane and glass. When all the above-mentioned requirements were completed, the success rate of gigaohm seal formation increased to about 90%. Although the stability of various patches varied, it was possible to achieve successful gigaseal contacts within 5 hours after obtaining the platelets.

Cell-attached mode for platelets

Due to the small size of platelets and the above-mentioned difficulties only a few ion channels in platelet membrane, including K_Ca 3.1, K_V 1.3, P2X, chloride channels, were identified and characterized using the patch-clamp technique. However, recent evidence

[

23

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Wright, J. R., Amisten, S., Goodall, A. H., & Mahaut-Smith, M. P.

Thrombosis and haemostasis. 2016, 116(2), 272

https://doi.org/10.1160/th15-11-0891

Article | Google Scholar

]

suggests that despite its small size and absence of nuclei, platelets possess a great variety of ion channels that are poorly characterized. Besides, previously measured experimental results were obtained using megakaryocyte, but not platelets. Therefore identification and characterization of ion channels in platelet membrane are of great importance for extending our knowledge about their role in various cell signaling pathways and physiological response

[

22,

Direct voltage control of signaling via P2Y1 and other Gαq-coupled receptors

Martinez-Pinna, J., Gurung, I. S., Vial, C., Leon, C., Gachet, C., Evans, R. J., & Mahaut-Smith, M. P.

Journal of Biological Chemistry. 2005, 280(2), 1490-1498

https://doi.org/10.1074/jbc.M407783200

Article | Google Scholar

30,

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel.

Martins, J. R., Faria, D., Kongsuphol, P., Reisch, B., Schreiber, R., & Kunzelmann, K.

Proceedings of the National Academy of Sciences. 2011, 108(44), 18168-18172

https://doi.org/10.1073/pnas.1108094108

Article | Google Scholar

31,

A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

Taylor, K. A., & Mahaut-Smith, M. P.

Platelets. 2019, 30(8), 962-966

https://doi.org/10.1080/09537104.2019.1595560

Article | Google Scholar

32,

Capacitative and non-capacitative signaling complexes in human platelets

Berna-Erro, A., Galan, C., Dionisio, N., Gomez, L. J., Salido, G. M., & Rosado, J. A.

Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2021, 1823(8), 1242-1251

https://doi.org/10.1016/j.bbamcr.2012.05.023

Article | Google Scholar

33,

Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity

Harper, M. T., & Poole, A. W.

Cell death & disease. 2013, 4(12), e969-e969

https://doi.org/10.1038/cddis.2013.495

Article | Google Scholar

34,

Regulation of STIM1/Orai1-dependent Ca2+ signalling in platelets.

Lang, F., Munzer, P., Gawaz, M., & Borst, O.

Thromb Haemost. 2013, 110(5), 925-930

https://doi.org/10.1160/th13-02-0176

Article | Google Scholar

35,

Transient receptor potential channels function as a coincidence signal detector mediating phosphatidylserine exposure

Harper, M. T., Londono, J. E. C., Quick, K., Londono, J. C., Flockerzi, V., Philipp, S. E., ... & Poole, A. W.

Science Signaling. 2013, 6(281), ra50-ra50

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

Article | Google Scholar

36

Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel

Wolfs, J. L., Wielders, S. J., Comfurius, P., Lindhout, T., Giddings, J. C., Zwaal, R. F., & Bevers, E. M.

Blood. 2006, 108(7), 2223-2228

https://doi.org/10.1182/blood-2006-01-009613

Article | Google Scholar

]

.

Measurement of single-channel ionic currents during platelet activation

Earlier it was shown that platelets possess a great amount (300-400 per cell) of voltage-gated potassium channels K_v 1.3

[

37

Kv1. 3 is the exclusive voltage‐gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count

McCloskey, C., Jones, S., Amisten, S., Snowden, R. T., Kaczmarek, L. K., Erlinge, D., ... & Mahaut‐Smith, M. P.

The Journal of physiology. 2010, 588(9), 1399-1406

https://doi.org/10.1113/jphysiol.2010.188136

Article | Google Scholar

]

. Considering the area of the platelet membrane and the size of the aperture of the patch pipette, at least a couple of these channels will always be present in the patch and will make a significant contribution to the registered ion channel currents, which will make it harder to distinguish other important ion channels. Taking into account the available data on the biophysical characteristics of this channel, namely the potential of half-activation of about -30 mV, we proposed the method to measure the activity of other ion channels. Usage of cell-attached configuration in voltage-clamp mode and application of a potential to patch pipette from +80 to +150 mV allowed us to avoid activation of voltage-gated ion channels and made it possible to register currents from other ion channels. During this procedure, the membrane under the patch pipette was hyperpolarized, which inactivated K_v 1.3 channels, at the same time there was enough driving force for all ions, which helped with the identification of even the smallest ionic currents in the range of 1-2 pS. Schematic illustration of the technique can be seen in fig. 1.

Schematic illustration of recording currents of single ion channels of platelets during activation, excluding K<sub>v</sub> 1.3

Figure 1. Schematic illustration of recording currents of single ion channels of platelets during activation, excluding K_v 1.3

Analysis of data from 6 patches allowed the identification of at least 5 different types of channels, with the conductivity varying from 2 to 20 pS. Considering that most of the identified and known channels have such a small conductivity, recordings with small applied potentials give the little result. When the applied potential is +50 mV, the resulting single-channel events have amplitudes of 0.4 – 0.5 pA, which only slightly exceeds the noise level (Fig. 2A). So we can recommend using potentials in the range of +80 – +150 mV. In that way, currents from even the smallest-conductance channels will be easily registered. Yet, high applied potentials may lead to relatively quick deterioration of the seal.

In fig. 2B the same recordings but with NaCl-based pipette BS can be seen. Regarding the observed amplitudes in NaCl salts, we obtained a much lower number of channels than in KCl saline, suggesting the involvement of more than 2 types of potassium channels in the process of activation of platelets by thrombin.

To test the stability of the patches obtained by the proposed method, we recorded single-channel ion currents from platelets in response to 1 μM of ionomycin, Ca²⁺-ionophore which induces strong platelet activation. Results of recordings of single-channel events with different pipette BS solutions are shown in fig. 2C.

A substantial rise in intracellular calcium levels, induced by ionomycin or combined action of collagen and thrombin, usually switches platelets to a new procoagulant state

[

38

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Schoenwaelder, S. M., Yuan, Y., Josefsson, E. C., White, M. J., Yao, Y., Mason, K. D., ... & Jackson, S. P.

Blood, The Journal of the American Society of Hematology. 2009, 114(3), 663-666

https://doi.org/10.1182/blood-2009-01-200345

Article | Google Scholar

]

. During this transition, the morphology of the cells changes drastically. They become balloon-shaped

[

38

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Schoenwaelder, S. M., Yuan, Y., Josefsson, E. C., White, M. J., Yao, Y., Mason, K. D., ... & Jackson, S. P.

Blood, The Journal of the American Society of Hematology. 2009, 114(3), 663-666

https://doi.org/10.1182/blood-2009-01-200345

Article | Google Scholar

]

, many of the plasma membrane proteins and phosphatidylserine move to the so-called cap region

[

40,

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

Podoplelova, N. A., Sveshnikova, A. N., Kotova, Y. N., Eckly, A., Receveur, N., Nechipurenko, D. Y., ... & Panteleev, M. A.

Blood, The Journal of the American Society of Hematology. 2016, 128(13), 1745-1755

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

Article | Google Scholar

41

Procoagulant platelets form an α-granule protein-covered “cap” on their surface that promotes their attachment to aggregates

Abaeva, A. A., Canault, M., Kotova, Y. N., Obydennyy, S. I., Yakimenko, A. O., Podoplelova, N. A., ... & Panteleev, M. A.

Journal of Biological Chemistry. 2013, 288(41), 29621-29632

https://doi.org/10.1074/jbc.M113.474163

Article | Google Scholar

]

. In Supplementary material, fig. S1 and fig S2, patched and floating platelets can be seen before and after this transition. Despite all that, only about 30% of patches deteriorated after activation with ionomycin.

Figure 2. Single channel events recorded from platelets in cell-attached mode. A, B

Ion channels play a pivotal role in a variety of processes underlying platelet activation. For example, potassium channels take part in calcium signaling, maintaining the membrane potential and thus increasing the driving force for Ca²⁺. Calcium entry during activation also depends on the functioning of many ion channels, including Orai1, P2X, TRPC

[

17,

Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes

Tolhurst, G., Carter, R. N., Amisten, S., Holdich, J. P., Erlinge, D., & Mahaut-Smith, M. P.

Platelets. 2008, 19(4), 308-313

https://doi.org/10.1080/09537100801935710

Article | Google Scholar

27,

Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte

Carter, R. N., Tolhurst, G., Walmsley, G., Vizuete‐Forster, M., Miller, N., & Mahaut‐Smith, M. P.

The Journal of physiology. 2006, 576(1), 151-162

https://doi.org/10.1113/jphysiol.2006.113886

Article | Google Scholar

42

Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets

MacKenzie, A. B., Mahaut-Smith, M. P., & Sage, S. O.

Journal of Biological Chemistry. 1996, 271(6), 2879-2881

https://doi.org/10.1074/jbc.271.6.2879

Article | Google Scholar

]

. Scramblase Ano6, which plays a pivotal role in platelet procoagulant response, also acts as an ion channel with chloride conductivity and has a very high threshold for activation by calcium

[

33,

Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity

Harper, M. T., & Poole, A. W.

Cell death & disease. 2013, 4(12), e969-e969

https://doi.org/10.1038/cddis.2013.495

Article | Google Scholar

43

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Fujii, T., Sakata, A., Nishimura, S., Eto, K., & Nagata, S.

Proceedings of the National Academy of Sciences. 2015, 112(41), 12800-12805

https://doi.org/10.1073/pnas.1516594112

Article | Google Scholar

]

. We believe that the proposed approach of registering single-channel currents is a useful tool to establish and investigate various platelet signaling events in which ion channels may play a key role.

Measurement of membrane potential in the cell-attached mode.

Previously, in the work by Mason J. et. al.

[

19

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

Mason, M. J., Simpson, A. K., Mahaut-Smith, M. P., & Robinson, H. P. C.

Biophysical journal. 2005, 88(1), 739-750

https://doi.org/10.1529/biophysj.104.049866

Article | Google Scholar

]

, a new method for studying membrane potential in a cell-attached model was proposed. According to the model experiments and patch-clamp data, the accuracy of the recorded potential in current-clamp depends on the ratio between seal resistance of the patch and input resistance of the membrane, as well as on the composition of the pipette solution, as it should mimic the intracellular composition of the cell. This method enables one to assess the membrane potential of platelets or any other cell type without breaking the membrane as for whole-cell configuration, in which fast perfusion of the intracellular constituents with electrode saline occurs.

To test this method for platelets we recorded the kinetics of membrane potential of platelets activated by human myeloperoxidase (MPO), which was shown previously to modulate platelet aggregation, cytoskeleton reorganization, and store-operated calcium entry

[

44

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

Gorudko, I. V., Sokolov, A. V., Shamova, E. V., Grudinina, N. A., Drozd, E. S., Shishlo, L. M., ... & Panasenko, O. M.

Biology open. 2013, 2(9), 916-923

https://doi.org/10.1242/bio.20135314

Article | Google Scholar

]

, as well as to activate neutrophils

[

45

Neutrophil activation in response to monomeric myeloperoxidase

Gorudko, I. V., Grigorieva, D. V., Sokolov, A. V., Shamova, E. V., Kostevich, V. A., Kudryavtsev, I. V., ... & Panasenko, O. M.

Biochemistry and Cell Biology. 2018, 96(5), 592-601

https://doi.org/10.1139/bcb-2017-0290

Article | Google Scholar

]

and to bind to human red blood cells and change their properties

[

46,

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells

Shamova, E. V., Gorudko, I. V., Grigorieva, D. V., Sokolov, A. V., Kokhan, A. U., Melnikova, G. B., ... & Panasenko, O. M.

Molecular and cellular biochemistry. 2020, 464(1), 119-130

https://doi.org/10.1007/s11010-019-03654-0

Article | Google Scholar

47

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level

Gorudko, I. V., Sokolov, A. V., Shamova, E. V., Grigorieva, D. V., Mironova, E. V., Kudryavtsev, I. V., ... & Timoshenko, A. V.

Archives of biochemistry and biophysics. 2016, 591, 87-97

https://doi.org/10.1016/j.abb.2015.12.007

Article | Google Scholar

]

. Adding 100 nM of MPO to extracellular bath solution led to a slight hyperpolarization of the plasma membrane at the level of 10-12 mV (fig. 3A).

Figure 3. Changes in plasma membrane potential of platelets

In some experiments, we observed strange spikes in recordings of potential in the cell-attached configuration. This effect can be seen in fig. 3B. These spikes were very rapid and had an amplitude of about 7-10 mV, suggesting that either these spikes arose from the changes of platelet membrane potential itself, or that openings of ion channels were involved.

We tried to investigate this behavior with a simple model. It is known that the input resistance of the membrane of platelets is around 50 GΩ

[

10

A patch‐clamp study of mammalian platelets and their voltage‐gated potassium current.

Maruyama, Y.

The Journal of physiology. 1987, None, None

https://doi.org/10.1113/jphysiol.1987.sp016750

Article | Google Scholar

]

. We’ve used the proposed simplified model

[

19

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

Mason, M. J., Simpson, A. K., Mahaut-Smith, M. P., & Robinson, H. P. C.

Biophysical journal. 2005, 88(1), 739-750

https://doi.org/10.1529/biophysj.104.049866

Article | Google Scholar

]

of measuring membrane potential in cell-attached mode and modified it by including single-channel conductance (G_channel), imitating single ion channels under patch pipette. The resulting electrical circuit can be seen in fig. 3C.

Modified equations are as follows:

Model parameters: G_input - 17 pS (59 GΩ), G_input- 5 pS or 24 pS (corresponding to the obtained in present study single-channel conductance’s), G_seal – variable (corresponding with patch resistance (R_contact) of 20, 30 и 50 GΩ). V_membrane = -60 mV. F(t) – the function of time, that determines the process of channel opening. In our case, it is a simple Gaussian function (eq. 2) with the following parameters: a = 1, b = 3, c = 0.07

The results of modeling (fig. 3D) indicate that the opening of ion channels led to spikes in membrane potential. The amplitude of spikes is inversely proportional to the contact resistance and directly proportional to the conductance of the opened channels. In the case of a theoretically perfect gigaohm seal (seal resistance approaches infinity), we wouldn’t be able to see any spikes in recorded potential during channel openings. Similarly, in the case of very small input resistance (for example, the addition of nystatin to the patch pipette) there also wouldn’t be any effect.

Earlier the oscillatory nature of membrane potential recordings in the cell-attached configuration was shown by Mason J. et. al. from platelets activated by ADP

[

19

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

Mason, M. J., Simpson, A. K., Mahaut-Smith, M. P., & Robinson, H. P. C.

Biophysical journal. 2005, 88(1), 739-750

https://doi.org/10.1529/biophysj.104.049866

Article | Google Scholar

]

. The results of the present study indicate that the reason for oscillatory behavior may be the openings of single channels in the patch. This should be taken into account, especially considering that the membrane potential of megakaryocytes can oscillate

[

20

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes

Mason, M. J., Hussain, J. F., & Mahaut-Smith, M. P.

The Journal of Physiology. 2000, 524(Pt 2), 437

https://doi.org/10.1111/j.1469-7793.2000.00437.x

Article | Google Scholar

]

.

Inside-out mode

The inside-out configuration is a useful tool that allows to control the ionic content of solutions on both sides of the patch membrane and to study the activation of ion channels by the direct action of secondary messengers in the absence of various intracellular signaling events.

To get this configuration, we used the method suggested earlier

[

48

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

MacKenzie, A. B., & Mahaut-Smith, M. P.

Biochimica et Biophysica Acta (BBA)-Biomembranes. 1996, 1278(1), 131-136

https://doi.org/10.1016/0005-2736(95)00207-3

Article | Google Scholar

]

. Briefly, a drop of viscous substance (we used immersion oil for microscopy) is placed on the bottom of the plate, after which the procedure of gigaohm seal formation as described earlier was performed. Then the pipette with the cell on its tip is brought to the edge of the oil drop and the cell is gently dipped into the oil. After contact between platelet and oil occurs, the tip is retracted to break off a patch of membrane from the rest of the cell, which remains in the oil. Following the above-mentioned recommendations we managed to obtain a successful inside-out patch in more than 90 % of cases, and it was stable for up to 10 min. Animation of the process of obtaining inside-out configuration can be seen in Supplementary materials, in fig. S3.

Figure 4. Single channel ionic currents recorded from platelets in inside-out configuration

Conclusions

At the moment patch-clamp method is the most powerful instrument for studying ion channels and their role in cell lifespan. During the past couple of decades ion channels, alongside numerous membrane receptors and intracellular messengers, have been realized to be an important constituent of the mechanisms of platelet activation and their physiological response. The present study provides the techniques and approaches that can help other researchers to extend the usage of the patch-clamp technique for the study of small cells and help to reveal new ways by which ion channels govern platelet functions.

Authors' contributions

A.U.K. performed experiments, analyzed the data, wrote the text, and edited the paper; S.O.Z. and I.I.P performed experiments, analyzed the data and edited the paper; I.V.G. analyzed the data and edited the paper; E.V.S. supervised the project, planned the research, analyzed the data and edited the paper.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank F. Balabin for the constructive discussion of experimental results and for editing the paper. The authors are grateful to Dr. Alexey Sokolov (Institute of Experimental Medicine, St. Petersburg, Russia) for the generously provided MPO and thrombin.

This work was partly supported by Belarusian Republican Foundation for Fundamental Research (grant B20R-215).

References of this article:

Dye indicators of membrane potential
Waggoner, A. S.
Annual review of biophysics and bioengineering. 1979, 8(1), 47-68
https://doi.org/10.1146/annurev.bb.08.060179.000403
Article|Google Scholar
Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes
Felix, J. P., Williams, B. S., Priest, B. T., Brochu, R. M., Dick, I. E., Warren, V. A., ... & Garcia, M. L.
Assay and drug development technologies. 2004, 2(3), 260-268
https://doi.org/10.1089/1540658041410696
Article|Google Scholar
Nitrate-selective optical sensor applying a lipophilic fluorescent potential-sensitive dye
Huber, C., Klimant, I., Krause, C., Werner, T., & Wolfbeis, O. S.
Analytica chimica acta. 2001, 449(1-2), 81-93
https://doi.org/10.1016/S0003-2670(01)01363-0
Article|Google Scholar
A Ca-dependent K channel in “luminal” membranes from the renal outer medulla
Burnham, C., Braw, R., & Karlish, S. J. D.
The Journal of membrane biology. 1986, 93(2), 177-186
https://doi.org/10.1007/BF01870809
Article|Google Scholar
A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles
Garty, H., Rudy, B., & Karlish, S. J.
Journal of Biological Chemistry. 1983, 258(21), 13094-13099
https://doi.org/10.1016/S0021-9258(17)44085-3
Article|Google Scholar
Amiloride blockable sodium fluxes in toad bladder membrane vesicles
Garty, H.
The Journal of membrane biology. 1984, 82(3), 269-279
https://doi.org/10.1007/BF01871636
Article|Google Scholar
Identification and reconstitution of a Na+/K+/Cl− cotransporter and K+ channel from luminal membranes of renal red outer medulla
Burnham, C., Karlish, S. J. D., & Jørgensen, P. L.
Biochimica et Biophysica Acta (BBA)-Biomembranes. 1985, 821(3), 461-469
https://doi.org/10.1016/0005-2736(85)90051-3
Article|Google Scholar
The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes
Neher, E., Sakmann, B., & Steinbach, J. H.
Pflügers Archiv. 1978, 375(2), 219-228
https://doi.org/10.1007/BF00584247
Article|Google Scholar
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
Hamill, O. P., Marty, A., Neher, E., Sakmann, B., & Sigworth, F. J.
Pflügers Archiv. 1981, 391(2), 85-100
https://doi.org/10.1007/BF00656997
Article|Google Scholar
A patch‐clamp study of mammalian platelets and their voltage‐gated potassium current.
Maruyama, Y.
The Journal of physiology. 1987, ,
https://doi.org/10.1113/jphysiol.1987.sp016750
Article|Google Scholar
Voltage‐gated potassium channels and the control of membrane potential in human platelets
Mahaut‐Smith, M. P., Rink, T. J., Collins, S. C., & Sage, S. O.
The Journal of physiology. 1990, 428(1), 723-735
https://doi.org/10.1113/jphysiol.1990.sp018237
Article|Google Scholar
Calcium‐activated potassium channels in human platelets.
Mahaut-Smith, M. P.
The Journal of physiology. 1995, 484(1), 15-24
https://doi.org/10.1113/jphysiol.1995.sp020644
Article|Google Scholar
Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique
Mahaut-Smith, M. P., Sage, S. O., & Rink, T. J.
Journal of Biological Chemistry. 1992, 267(5), 3060-3065
https://doi.org/10.1016/S0021-9258(19)50694-9
Article|Google Scholar
Receptor-activated single channels in intact human platelets
Mahaut-Smith, M. P., Sage, S. O., & Rink, T. J.
Journal of Biological Chemistry. 1990, 265(18), 10479-10483
https://doi.org/10.1016/S0021-9258(18)86972-1
Article|Google Scholar
Three cation influx currents activated by purinergic receptor stimulation in rat megakaryocytes
Somasundaram, B., & Mahaut-Smith, M. P.
The Journal of physiology. 1994, 480(2), 225-231
https://doi.org/10.1113/jphysiol.1994.sp020355
Article|Google Scholar
Primaquine, an inhibitor of vesicular transport, blocks the calcium-release-activated current in rat megakaryocytes
Somasundaram, B., Norman, J. C., & Mahaut-Smith, M. P.
Biochemical Journal. 1995, 309(3), 725-729
https://doi.org/10.1042/bj3090725
Article|Google Scholar
Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes
Tolhurst, G., Carter, R. N., Amisten, S., Holdich, J. P., Erlinge, D., & Mahaut-Smith, M. P.
Platelets. 2008, 19(4), 308-313
https://doi.org/10.1080/09537100801935710
Article|Google Scholar
Probes of transmembrane potentials in platelets: changes in cyanine dye fluorescence in response to aggregation stimuli.
Home, W. C., & Simons, E. R.
Blood. 1978, 51(4), 741-749
https://doi.org/10.1182/blood.V51.4.741.741
Article|Google Scholar
The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration
Mason, M. J., Simpson, A. K., Mahaut-Smith, M. P., & Robinson, H. P. C.
Biophysical journal. 2005, 88(1), 739-750
https://doi.org/10.1529/biophysj.104.049866
Article|Google Scholar
A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes
Mason, M. J., Hussain, J. F., & Mahaut-Smith, M. P.
The Journal of Physiology. 2000, 524(Pt 2), 437
https://doi.org/10.1111/j.1469-7793.2000.00437.x
Article|Google Scholar
The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling
Rinne, A., Mobarec, J. C., Mahaut-Smith, M., Kolb, P., & Bünemann, M.
Science Signaling. 2015, 8(401), ra110-ra110
https://doi.org/10.1126/scisignal.aac7419
Article|Google Scholar
Direct voltage control of signaling via P2Y1 and other Gαq-coupled receptors
Martinez-Pinna, J., Gurung, I. S., Vial, C., Leon, C., Gachet, C., Evans, R. J., & Mahaut-Smith, M. P.
Journal of Biological Chemistry. 2005, 280(2), 1490-1498
https://doi.org/10.1074/jbc.M407783200
Article|Google Scholar
Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.
Wright, J. R., Amisten, S., Goodall, A. H., & Mahaut-Smith, M. P.
Thrombosis and haemostasis. 2016, 116(2), 272
https://doi.org/10.1160/th15-11-0891
Article|Google Scholar
Expression and functional characterization of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 in megakaryocytes and platelets
Balduini, A., Fava, C., Di Buduo, C. A., Abbonante, V., Meneguzzi, A., Soprano, P. M., ... & Minuz, P.
J Thromb Haemost. 2021, ,
https://doi.org/10.1111/jth.15269
Article|Google Scholar
Patch-clamp recordings of electrophysiological events in the platelet and megakaryocyte.
Mahaut-Smith, M. P.
In Platelets and Megakaryocytes . 2004, , 277-299
https://doi.org/10.1385/1-59259-783-1:277
Article|Google Scholar
Interplay between P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling
Tolhurst, G., Vial, C., Léon, C., Gachet, C., Evans, R. J., & Mahaut-Smith, M. P.
Blood. 2005, 106(5), 1644-1651
https://doi.org/10.1182/blood-2005-02-0725
Article|Google Scholar
Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte
Carter, R. N., Tolhurst, G., Walmsley, G., Vizuete‐Forster, M., Miller, N., & Mahaut‐Smith, M. P.
The Journal of physiology. 2006, 576(1), 151-162
https://doi.org/10.1113/jphysiol.2006.113886
Article|Google Scholar
Patch clamp techniques for single channel and whole-cell recording
Ogden, D., & Stanfield, P.
In Microelectrode techniques: the Plymouth workshop handbook . 1994, , 53-78
Google Scholar
Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording
Priel, A., Gil, Z., Moy, V. T., Magleby, K. L., & Silberberg, S. D.
Biophysical journal. 2007, 92(11), 3893-3900
https://doi.org/10.1529/biophysj.106.099119
Article|Google Scholar
Anoctamin 6 is an essential component of the outwardly rectifying chloride channel.
Martins, J. R., Faria, D., Kongsuphol, P., Reisch, B., Schreiber, R., & Kunzelmann, K.
Proceedings of the National Academy of Sciences. 2011, 108(44), 18168-18172
https://doi.org/10.1073/pnas.1108094108
Article|Google Scholar
A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01
Taylor, K. A., & Mahaut-Smith, M. P.
Platelets. 2019, 30(8), 962-966
https://doi.org/10.1080/09537104.2019.1595560
Article|Google Scholar
Capacitative and non-capacitative signaling complexes in human platelets
Berna-Erro, A., Galan, C., Dionisio, N., Gomez, L. J., Salido, G. M., & Rosado, J. A.
Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2021, 1823(8), 1242-1251
https://doi.org/10.1016/j.bbamcr.2012.05.023
Article|Google Scholar
Chloride channels are necessary for full platelet phosphatidylserine exposure and procoagulant activity
Harper, M. T., & Poole, A. W.
Cell death & disease. 2013, 4(12), e969-e969
https://doi.org/10.1038/cddis.2013.495
Article|Google Scholar
Regulation of STIM1/Orai1-dependent Ca2+ signalling in platelets.
Lang, F., Munzer, P., Gawaz, M., & Borst, O.
Thromb Haemost. 2013, 110(5), 925-930
https://doi.org/10.1160/th13-02-0176
Article|Google Scholar
Transient receptor potential channels function as a coincidence signal detector mediating phosphatidylserine exposure
Harper, M. T., Londono, J. E. C., Quick, K., Londono, J. C., Flockerzi, V., Philipp, S. E., ... & Poole, A. W.
Science Signaling. 2013, 6(281), ra50-ra50
https://doi.org/10.1126/scisignal.2003701
Article|Google Scholar
Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel
Wolfs, J. L., Wielders, S. J., Comfurius, P., Lindhout, T., Giddings, J. C., Zwaal, R. F., & Bevers, E. M.
Blood. 2006, 108(7), 2223-2228
https://doi.org/10.1182/blood-2006-01-009613
Article|Google Scholar
Kv1. 3 is the exclusive voltage‐gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count
McCloskey, C., Jones, S., Amisten, S., Snowden, R. T., Kaczmarek, L. K., Erlinge, D., ... & Mahaut‐Smith, M. P.
The Journal of physiology. 2010, 588(9), 1399-1406
https://doi.org/10.1113/jphysiol.2010.188136
Article|Google Scholar
Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function
Schoenwaelder, S. M., Yuan, Y., Josefsson, E. C., White, M. J., Yao, Y., Mason, K. D., ... & Jackson, S. P.
Blood, The Journal of the American Society of Hematology. 2009, 114(3), 663-666
https://doi.org/10.1182/blood-2009-01-200345
Article|Google Scholar
Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy
Hess, M. W., & Siljander, P.
Histochemistry and cell biology. 2001, 115(5), 439-443
https://doi.org/10.1007/s004180100272
Article|Google Scholar
Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting
Podoplelova, N. A., Sveshnikova, A. N., Kotova, Y. N., Eckly, A., Receveur, N., Nechipurenko, D. Y., ... & Panteleev, M. A.
Blood, The Journal of the American Society of Hematology. 2016, 128(13), 1745-1755
https://doi.org/10.1182/blood-2016-02-696898
Article|Google Scholar
Procoagulant platelets form an α-granule protein-covered “cap” on their surface that promotes their attachment to aggregates
Abaeva, A. A., Canault, M., Kotova, Y. N., Obydennyy, S. I., Yakimenko, A. O., Podoplelova, N. A., ... & Panteleev, M. A.
Journal of Biological Chemistry. 2013, 288(41), 29621-29632
https://doi.org/10.1074/jbc.M113.474163
Article|Google Scholar
Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets
MacKenzie, A. B., Mahaut-Smith, M. P., & Sage, S. O.
Journal of Biological Chemistry. 1996, 271(6), 2879-2881
https://doi.org/10.1074/jbc.271.6.2879
Article|Google Scholar
TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets
Fujii, T., Sakata, A., Nishimura, S., Eto, K., & Nagata, S.
Proceedings of the National Academy of Sciences. 2015, 112(41), 12800-12805
https://doi.org/10.1073/pnas.1516594112
Article|Google Scholar
Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry
Gorudko, I. V., Sokolov, A. V., Shamova, E. V., Grudinina, N. A., Drozd, E. S., Shishlo, L. M., ... & Panasenko, O. M.
Biology open. 2013, 2(9), 916-923
https://doi.org/10.1242/bio.20135314
Article|Google Scholar
Neutrophil activation in response to monomeric myeloperoxidase
Gorudko, I. V., Grigorieva, D. V., Sokolov, A. V., Shamova, E. V., Kostevich, V. A., Kudryavtsev, I. V., ... & Panasenko, O. M.
Biochemistry and Cell Biology. 2018, 96(5), 592-601
https://doi.org/10.1139/bcb-2017-0290
Article|Google Scholar
The effect of myeloperoxidase isoforms on biophysical properties of red blood cells
Shamova, E. V., Gorudko, I. V., Grigorieva, D. V., Sokolov, A. V., Kokhan, A. U., Melnikova, G. B., ... & Panasenko, O. M.
Molecular and cellular biochemistry. 2020, 464(1), 119-130
https://doi.org/10.1007/s11010-019-03654-0
Article|Google Scholar
Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level
Gorudko, I. V., Sokolov, A. V., Shamova, E. V., Grigorieva, D. V., Mironova, E. V., Kudryavtsev, I. V., ... & Timoshenko, A. V.
Archives of biochemistry and biophysics. 2016, 591, 87-97
https://doi.org/10.1016/j.abb.2015.12.007
Article|Google Scholar
Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium
MacKenzie, A. B., & Mahaut-Smith, M. P.
Biochimica et Biophysica Acta (BBA)-Biomembranes. 1996, 1278(1), 131-136
https://doi.org/10.1016/0005-2736(95)00207-3
Article|Google Scholar