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Keywords:

  • calcium;
  • connexins;
  • ion channels;
  • ionotropic glutamate receptors;
  • K+ channels;
  • megakaryocytes;
  • membrane potential;
  • Orai1;
  • P2X1;
  • platelets

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Summary.  Ion channels are transmembrane proteins that play ubiquitous roles in cellular homeostasis and activation. In addition to their recognized role in the regulation of ionic permeability and thus membrane potential, some channel proteins possess intrinsic kinase activity, directly interact with integrins or are permeable to molecules up to ≈1000 Da. The small size and anuclear nature of the platelet has often hindered progress in understanding the role of specific ion channels in hemostasis, thrombosis and other platelet-dependent events. However, with the aid of transgenic mice and ‘surrogate’ patch clamp recordings from primary megakaryocytes, important unique contributions to platelet function have been identified for several classes of ion channel. Examples include ATP-gated P2X1 channels, Orai1 store-operated Ca2+ channels, voltage-gated Kv1.3 channels, AMPA and kainate glutamate receptors and connexin gap junction channels. Furthermore, evidence exists that some ion channels, such as NMDA glutamate receptors, contribute to megakaryocyte development. This review examines the evidence for expression of a range of ion channels in the platelet and its progenitor cell, and highlights the distinct roles that these proteins may play in health and disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Ion channels have well-established roles in cellular responses via their ability to regulate membrane ionic permeability. This allows control of membrane potential, cell volume and intracellular ion concentrations, including levels of the ubiquitous second messenger Ca2+. Evidence is also emerging for more novel mechanisms whereby ion channels may alter cellular responses, including intrinsic kinase activity [1,2], coupling of channel voltage sensors to phosphatases [3] and direct interactions with integrins [4,5]. Like any platelet protein, an ion channel may influence the physiological processes of hemostasis and tissue repair, or may impact on the morbidity and mortality resulting from arterial thrombosis. In addition, it is now recognized that platelets contribute to several other physiological and pathophysiological processes, including liver regeneration, atherosclerosis, asthma, cancer and angiogenesis [6]. Finally, it should also be considered that platelet proteins may serve no crucial function in these anuclear blood cells but simply represent the vestigial remains of expression in the megakaryocyte after contributing to megakaryopoiesis and thrombopoiesis. This review discusses the evidence for different classes of ion channel in the platelet and megakaryocyte and their proposed roles to date (summarized in Fig. 1 and Table S1). There is now substantial evidence for a functional role of several types of Ca2+ -permeable ion channel in platelet activation, particularly P2X1 cation channels, Orai1 store-operated channels and organellar IP3 receptors. More than 20 further types of ion channel, with a range of ionic selectivity, have been reported to be active in the platelet or megakaryocyte, although for many of these the evidence for a contribution to cellular function relies upon a small number of studies. The gold standard for measuring ion channel activity in single cells is the patch clamp technique, which has proven difficult to apply to the small and fragile platelet. Consequently, megakaryocyte electrophysiological recordings have often served as a substitute for the platelet, justified by the observation that the mature progenitor cell expresses platelet-specific signaling pathways and functional responses [7]. My apologies to those authors whose work I have been unable to cite owing to space limitations. Please also note that this review focuses on ion channels for which there is evidence of a contribution to platelet and megakaryocyte function beyond detection by microarray or proteomics approaches.

image

Figure 1.  Summary of ion channels reported in the platelet or megakaryocyte. The membrane potential (Em) is reported to be approximately −50 mV at rest (set mainly by Kv1.3) and to hyperpolarize to approximately −80 mV in the early stages of platelet activation (set mainly by KCa3.1), but may depolarize later owing to opening of choride channels, non-selective cation channels (e.g. AMPA, P2X1, TRPs) or larger pore connexin gap junction channels and hemichannels (latter not shown). The direction of ionic flux depicted by the arrows is for Em in the range −50 to −80 mV. Cytosolic activation signals are encoded blue. TKRs, tyrosine kinase-linked receptors; GPCRs, G-protein-coupled receptors; DTS, dense tubular system. See Table S1 and text for explanation of other abbreviations.

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P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

P2X receptors are non-selective cation channels whose principal, and probably exclusive physiological agonist, is ATP [8]. Of the seven P2X receptor subunits (P2X1–P2X7) identified in mammals, only P2X1 receptors are expressed at significant levels in platelets and megakaryocytes [9,10]. P2X1 channels are directly gated by ATP binding and thus represent the fastest mechanism whereby tissue damage can lead to an increase in the important second messenger Ca2+ within the platelet. It is also important to note that ATP does not activate P2Y1 and P2Y12 receptor responses at the low expression levels of these ADP-stimulated GPCRs in platelets [11]. Therefore, P2X1 receptors represent the only known mechanism whereby the elevations of ATP that occur after vascular injury [12] can directly stimulate platelets. Selective activation of P2X1 receptors in vitro evokes a rapid and transient Ca2+ and Na+ influx, shape change, centralization of dense granules and minor levels of aggregation [13–15]. In addition, P2X1 receptors play important roles in the aggregation and secretion responses to low or intermediate concentrations of collagen [13,16] and low levels of protease activated receptor (PAR) activation [17]. Efficient local activation of P2X1 and Ca2+ influx by secreted ATP probably contributes to these key amplifying roles of P2X1 [18,19]. Synergistic interactions of ATP-gated P2X1 with receptors for adrenaline, thrombin/adrenaline and thrombopoietin have also been reported [20]. The direct or amplifying effects of P2X1 depend upon Ca2+ influx, with no evidence for a contribution by the Na+ entry or depolarization also evoked by channel opening. In vitro, under conditions of flow, the relative contribution of platelet P2X1 receptors to thrombus formation over collagen-coated surfaces increases with the shear rate [13,16]. This can account for the greater contribution of P2X1 receptors to arterial compared with venous thrombosis [11,13,21]. A further important property of P2X1 receptors is their resistance to inhibition by cyclic nucleotides, and thus several endogenous platelet inhibitory systems (e.g. prostacyclin and nitric oxide) [19]. ATP secretion evoked by collagen-stimulated GPVI and Toll-like receptors is also resistant to cyclicAMP and cyclicGMP [19], thus P2X1 channels represent a particularly important route for elevation of platelet [Ca2+]i during the early stages of hemostasis or in the intact circulation when innate immune responses are stimulated. For additional discussion of P2X1 receptors and their contribution to function in the platelet, readers are directed to a recent comprehensive review [11].

Ligand-gated cation channels activated by neurotransmitters other than ATP

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

In addition to ATP, platelets secrete several major neurotransmitters, including serotonin, glutamate and acetylcholine [22]. There is evidence for all three types of ionotropic glutamate receptor (NMDA, AMPA and kainate, named after their selective agonists) in the platelet and/or megakaryocyte. Morrell et al. have described AMPA and kainate receptors in human and murine platelets [23,24] and used real-time amperometry to demonstrate peak glutamate levels in excess of 400 μm in whole blood after thrombin stimulation [24]. In mice, kainate and AMPA receptor antagonists prolong tail bleed times and reduce FeCl3-induced thrombus development. In vitro, glutamate on its own does not stimulate functional responses such as inside-out activation of αIIbβ3, but glutamate, kainate or AMPA enhance responses to classic GPCR agonists such as thromboxane A2 and thrombin receptor-activating peptide (TRAP) [23,24]. In addition, TRAP-induced platelet activation is inhibited by the AMPA antagonist CNQX, suggesting a role for autocrine/paracrine activation by secreted glutamate. Four different AMPA subtypes exist, GluA1–GluA4, also known as GluR1–GluR4, which form functional channels as homo- or hetero-tetrameric assemblies. Platelets express all four AMPA receptor subtypes and small AMPA-activated currents were observed in whole-cell patch clamp recordings from murine megakaryocytes [24]. GluA1-deficient mice show resistance to thrombosis, suggesting an important role for this subtype in platelet AMPA channels [24]. Most AMPA channels are monovalent cation channels as they include the GluA2 subunit, although GluA2-lacking Ca2+ -permeable AMPA channels have been demonstrated in many areas of the brain [25]. A linear current–voltage relationship and lack of effect of the toxin Joro-2 suggest that megakaryocytes, and thus platelets, express GluA2-containing Ca2+ -impermeable AMPA receptors [24,26]. Kainate also activates monovalent cationic currents in murine megakaryocytes [23]. Five kainate receptor subunits have been described, known as GluK1-5 (previously referred to as GluR5-7, KA1 and KA2, respectively). Platelets express GluK1 (GluR5) and GluK2 (GluR6) and roles for both in the actions of kainate are suggested from experiments with a GluK1 agonist and GluK2-deficient mice. GluK2-deficient mice show resistance to FeCl3-induced arteriolar thrombosis and wild-type mice treated with a kainate receptor antagonist show prolonged bleeding. It is unclear why such small AMPA- and kainate-activated currents were observed (< 10 pA at −60 mV) given the large membrane capacitance of the megakaryocyte [27] and that a desensitization inhibitor, cyclothiazide, was required to unmask currents [24]. Spontaneous glutamate release and thus desensitization may be responsible, as observed for ATP release and P2X1 receptors [11]. Kainate stimulates thromboxane A2 generation via the p38 MAPK pathway and interestingly, an association with platelet responses after aspirin treatment was observed for GluK1 and 2 (GluR5 and 6) polymorphisms [23]. Exactly how kainate and AMPA channels potentiate platelet activation without generating Ca2+ influx is unclear. Na+ entry could lead to Ca2+ influx via the reverse mode of the Na+/Ca2+ exchanger; alternatively the depolarizing influence could potentiate GPCRs as shown in the megakaryocyte [28] or cytosolic Na+ may have novel direct roles. When glutamate is the ligand, kainate and AMPA receptors could depolarize the cell to open NMDA receptors (see below). It is particularly interesting to note that high blood glutamate levels have been observed after a stroke, thus AMPA/kainate channels may contribute to the increased risk of thrombosis in such patients (summarized in [24]).

NMDA receptor subunits fall into three distinct classes, GluN1, GluN2 and GluN3 (also known as NR1, NR2 and NR3). Four GluN2 (GluN2A-D) and two GluN3 (GluN3A,B) subtypes, together with multiple splice variants of GluN1, allow for significant diversity in channel properties. Most functional NMDA receptor ion channels are tetrameric assemblies of two obligatory GluN1 subunits together with two GluN2 subunits, but GluN3 subunits have been reported to substitute for the GluN2 subunits [22]. Glutamate binds to N2 subunits and the mandatory co-agonist glycine (or D-serine or D-alanine) binds to N1 and N3 subunits (thus glycine may activate the channels without glutamate when only GluN1 and 3 units are present). CD34+ -derived human megakaryocytes express GluN1 along with both GluN2A and GluN2D subunits [29], thus allowing for functional NMDA channels. GluN1 and GluN2D, but not GluN2A transcripts, were also found in Meg-01 cells and rat marrow [29]. The NMDA receptor antagonist MK-801 binds to native megakaryocytes, inhibits human megakaryocyte development from umbilical cord blood CD34+ cells, inhibits phorbol ester-induced megakaryocyte surface marker expression in the cell line Meg-01 and inhibits proplatelet formation from megakaryocytes without affecting cell survival [29,30]. In contrast, NMDA has been reported either to have no effect on platelet responses [23,24] or to inhibit platelet aggregation induced by several agonists [31,32], possibly via an in increase the inhibitory messenger cyclicAMP. However, in the early work of Franconi et al., glutamate was also reported to be inhibitory, which is inconsistent with later studies by the Morrell group [23,24]. Furthermore, a brief recent report suggests that NMDA receptor antagonists can inhibit platelet functional responses [33], thus further studies are required. NMDA receptors are non-selective cation channels with significant Ca2+ -permeability but at normal resting potentials the channels are blocked by resident Mg2+ ions such that depolarization is normally required to allow the receptor to generate Ca2+ influx [22]. NMDA has been shown to evoke only a small increase in [Ca2+]i in human platelets [32]; whether the Ca2+ response is limited by a relatively hyperpolarized membrane potential [34–38] or lack of the co-agonists glycine or D-serine in this study is unclear. It is worth noting that functional NMDA receptor channels in the platelet or megakaryocyte have only been inferred by binding of MK-801, a pore-blocker of the channel [39]; patch clamp recordings of NMDA receptor currents have not been reported.

A brief report using Western blotting and flow cytometry suggests the presence of serotonin-gated 5-HT3A subunits in the plasma membrane of human platelets and an increase in their surface expression after activation of PARs and P2Y receptors [40]. The presence of 5-HT3A subunits alone is sufficient to allow functional serotonin-gated non-selective cation channels [41]. Non-selective cation channels activated by serotonin and blocked by the 5-HT3 antagonist D-tubocurarine have been described in a whole cell patch clamp study of human platelets [42]. It remains unclear why the serotonin-gated inward currents had amplitudes several fold higher than any other agonist-evoked currents reported in the platelet, including P2X1 [43,44]. In addition, 5HT3A receptor channels are Ca2+ -permeable and to my knowledge no study of serotonin activation has reported Ca2+ mobilization independent of 5HT2 GPCRs. The functional role of platelet 5HT3A receptors is also unknown, but they could potentially contribute to responses after the release of serotonin from dense granules. A recent study has reported the presence of Ca2+ -permeable α7 nicotinic cholinergic receptors (nAChRα7) in platelets and the MEG01 cell line [45]. The Ca2+ increase develops only slowly, unlike classic neuronal cholinergic receptor currents, and is only small in amplitude although it declines slowly. As observed for stimulation of glutamate ionotropic receptors, cholinergic agonists do not cause platelet activation but can weakly potentiate responses to thromboxane A2 and ADP. The nAChRα7 antagonists methyllycaconitine and α-bungarotoxin also reduce platelet activation and phorbol ester-induced Meg-01 differentiation [45,46]. As noted above for NMDA receptors, nicotinic cholinergic-evoked cation currents have not been reported in the platelet or megakaryocyte.

Organellar ion channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

IP3 receptors are non-selective cation channels located on the membranes of organelles that form the intracellular Ca2+ store, which is located in the endoplasmic reticulum although in the platelet this is referred to as the dense tubular system. These channels are co-activated by cytosolic IP3 and Ca2+, with most studies also reporting that high Ca2+ becomes inhibitory (≥500 nm, but highly variable between studies [47]). Platelets have been shown to express all three types of IP3 receptors [48–50]. The relative importance of each subtype has not been clearly defined, although type I clearly plays a major role in release of internally stored Ca2+ [49–52]. Type II and III IP3 receptors have been suggested to reside on the plasma membrane [49,50], which could contribute to Ca2+ influx during activation; however, this has not been clearly demonstrated. IP3 infusion into the megakaryocyte cytoplasm generates a sustained or oscillatory inward cation current in whole-cell patch clamp recordings, but the identity of this conductance, its Ca2+ permeability and whether IP3 directly activates or merely induces the current remains unclear [53–55]. An important property of platelet IP3 receptors is their complete inhibition by protein kinases A or G, as this is a major mechanism by which endothelial-derived prostacyclin or nitric oxide maintain platelets in a resting condition in the intact circulation [56–58]. For PKG, this inhibition involves the protein IRAG [51], but for PKA, the mechanism is not clear.

Cation-permeable two-pore channels (TPC) have emerging roles in Ca2+ release from acidic organelles (lysosomes and lysosome-related organelles) in a variety of cells [59]. The proposed main ligand of TPC channels, NAADP, is generated by ADP ribosyl cyclases such as CD38 and represents the most potent known cytosolic signal for releasing Ca2+. Interestingly, CD38 deletion in mice reduces thrombin-evoked Ca2+ responses and phosphatidylserine exposure, prolongs bleed times and reduces thrombus stability [60]. Another CD38 product, cADPribose, may be co-involved in these responses; however, cADPribose targets the ryanodine receptor and no study has convincingly demonstrated that this Ca2+ -activated Ca2+ release channel is functional in the platelet or megakaryocyte. NAADP-induced Ca2+ increases have been described in permeabilized platelets; in addition, depletion of acidic stores with TBHQ or the TPC antagonist Ned19 reduces agonist-evoked Ca2+ increases [60–62]. Ned19 also inhibited platelet functional responses to various agonists in vitro [62]. As dense granules are lysosomal-related organelles [63], and platelet lysosomes are secretory vesicles, it is interesting to speculate that TPC channels may be inserted into the plasma membrane and facilitate Ca2+ entry after platelet activation. Two TPC isoforms exist, TPC1 and 2. TPC1 (also known as TPCN1) has been detected in a proteomics screen of platelets, and both isoforms detected in the Meg-01 cell line [64].

Platelets express the chloride intracellular channel 1 (CLIC1) which has a central punctate localization at rest, as expected for a vesicular distribution, but with some movement to the periphery upon exposure to ADP, which could indicate insertion into the plasma membrane upon activation [65]. CLIC1 has also been detected in a membrane proteomics screen [66]. The protein is unusual for an ion channel as both soluble and membrane-inserted forms exist. Murine CLIC1 deficiency is associated with a mild bleeding phenotype and reduced ADP-evoked activation, suggested to result from a change in P2Y12- but not P2Y1-dependent signaling [65].

Gap junctions and hemichannels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Effective communication between adjacent cells is important for co-ordinating a variety of physiological responses. When cells are in close physical contact, such as in the marrow or in a thrombus, direct intercellular communication via gap junction (GJ) channels becomes a possibility. In vertebrates, GJ channels are formed by the connexin protein family [67]. Hexameric connexin assemblies form hemichannels (also termed connexons), and coupling of connexons between adjacent cells forms GJs. Hemichannels and GJs are permeable to ions and small molecules up to ≈1000 Da. A number of reports dating back to 1979 have proposed a role for gap junction channels in intercellular communication between marrow cells, based on electron microscopy and the movement of dyes between cells (summarized in [68]). The studies conclude that connexin 43 is the major GJ-forming subunit expressed in hematopoietic tissue (summarized in [69]), and in megakaryocytes allows communication with stromal cells [70] and osteoblasts [71]. There is considerable evidence to support a role for connexin 43 in hematopoiesis [72,73], although perinatal lethality of the knock-out in mice has complicated studies. In an inducible gene-targeted murine model, connexin 43 deficiency does not alter basal blood cell or platelet counts, but does severely limit recovery from cytopenia induced by the chemotherapeutic drug 5-fluorouracil [74]. Connexin 43 deficiency appears to induce a maturation arrest at the level of long-term hematopoietic stem cells. Megakaryocytes have been shown to stimulate osteoblast proliferation through a form of direct coupling or juxtacrine mechanism, and the substantial increase in immature megakaryocytes observed in GATA-1−/− and NF-E2−/− mice is proposed to account for the phenotype of enhanced bone mass [75]. However, block of gap junctions enhances this proliferative effect of megakaryocytes, without an effect on osteoblast differentiation, thus the exact role of connexins in megakaryocyte–osteoblast interactions under normal conditions is unclear [71]. Two groups have recently reported connexin 37 in megakaryocytes [76,77], although platelet morphology and counts are normal in connexin 37-deficient mice. The two studies also report novel roles for connexin 37 in the platelet, but with very different results and conclusions. Evidence for a major contribution of this GJ protein to platelet activation is provided in one study where aggregation, clot retraction and laser-induced arterial thrombosis were all reduced upon application of connexin blockers or in connexin 37−/− mice [77]. Furthermore, fluorescence measurements show calcein diffusion between platelets within a thrombus and electron microscopy shows GJ-like structures between the membranes of aggregated platelets [77]. Responses in isolated platelets were also reduced by connexin blockers, thereby suggesting an involvement of hemi-channel activity [77]. In contrast, work from a separate group proposes an inhibitory role for connexin 37 in platelets, as application of inhibitors or connexin 37 deficiency led to reduced bleeding, increased aggregation and enhanced FeCl3-induced thrombosis or thromboembolism induced by collagen and epinephrine [76]. Further work is required to understand the differences between the two studies, the underlying mechanism(s) whereby GJs achieve interplatelet communication, and also the roles of other connexins detected in human platelets [77].

Voltage-gated K+ channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

The largest amplitude ionic currents recorded within patch clamp studies of platelets and mature megakaryocytes from a number of mammalian species are conducted through depolarization-gated K+-selective (Kv) channels [34,36,78]. They have a threshold for activation of approximately −60 mV, are steeply voltage-dependent in the range −40 to −10 mV and are maximally activated at ≥ 0 mV [34,36,79]. A recent study has demonstrated that a single alpha subunit, Kv1.3, encoded by KCNA3, is responsible for forming this channel in human platelets and murine megakaryocytes [37]. This conductance clearly sets the resting potential of approximately −50 mV but most likely also contributes to the membrane potential during agonist stimulation since blockade with margatoxin leads to a small reduction in agonist-evoked or store-operated Ca2+ entry [34,37]. Interestingly, Kv1.3-deficient mice have slightly elevated platelet counts, although the underlying mechanism is unknown. Kv1.3 channels play crucial roles in volume regulation and cell proliferation in lymphocytes [80,81], but these possibilities have not been investigated in the megakaryocyte and platelet. In one study of human megakaryocytes, the K+ current phenotype was observed to change with development, with small cells of ‘megakaryocytic’ lineage expressing only a voltage-gated inward rectifier or no current. However, further validation of this conclusion is required as an antibody against GPIIbIIIa (i.e. αIIbβ3) was used to identify megakaryocytes and yet mast cells and haematopoietic progenitors also express GPIIb [82]. Deletion of Kv1.3 has no gross effect on megakaryocyte size distribution in the mouse [37]. Of potential relevance to the platelet, Kv channels have been proposed to interact directly with β1 integrins to regulate lymphocyte adhesion [4,5]. This possibility requires further investigation using K+ channel-deficient murine models as experiments to date have depended upon elevated extracellular K+ to depolarize the lymphocytes and activate Kv channels. A role for G-protein-gated inwardly rectifying potassium channels in platelet P2Y12-coupled signaling has been described in one study [83], but the existence of such channels is not supported by electrophysiological recordings [34,36,37].

An interesting, and as yet unexplained, observation is that cell lines of megakaryoblastic (DAMI, CHRF-288-11) and erythroleukemic (HEL) origin lack depolarization-gated K+ currents [84,85]. Furthermore, this conductance was substantially reduced in a high percentage of primary megakaryocytes from patients with acute myelogenous leukemia (AML), but not those with acute lymphocytic leukemia [86]. Although only assessed in one patient, chemotherapy led to reappearance of this voltage-gated K+ conductance. It is unclear whether the lack of voltage-gated K+ currents contributes to the condition of AML. Of relevance, however, are reports that K+ channels, including Kv1.3, are involved in apoptosis [87,88].

Ca2+ -activated K+ channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

The first strong evidence for platelet Ca2+ -dependent K+ (KCa) channels came from fluorescent indicator measurements of hyperpolarizations induced by the Ca2+ ionophore A23187 [89]. Three classes of KCa channel exist, divided on the basis of their single channel conductance into small (SKCa), intermediate (known as KCa3.1, IKCa, the Gardos channel or KCa4, encoded by the gene KCNN4) and large (BKCa). Whole cell patch clamp recordings indicate that human platelets express only intermediate conductance KCa channels [90], which is supported by the ability of charybdotoxin (CTX), but not apamin (a SKCa inhibitor), to block Ca2+ -dependent hyperpolarizations [89]. The pharmacology and electrophysiology of KCa currents in megakaryocytes and related cell lines supports this conclusion (inwardly rectifying, single channel conductance of ≈24pS and block by CTX, clotrimazole and Ba2+ but not by apamin, iberiotoxin or 4-aminopyridine) [38,85,89,91,92]. Recently, immunofluorescence studies have suggested a high density of KCa3.1 channels in human and murine platelets [93], although direct patch clamp studies report only a small number (5–7 channels) per platelet [90]. This is much lower than the density of voltage-gated K+ channels (Kv1.3) estimated under similar whole-cell conditions (> 100 per platelet [34,36]). Nevertheless, a small number of KCa3.1 channels may play a significant role in determining the membrane potential, and thus Ca2+ influx, in stimulated platelets as they are half-maximally activated by a [Ca2+]i of ≈300 nm and show little voltage-dependence. In line with this role, oscillatory KCa activity in megakaryocytes causes repetitive hyperpolarizations from the resting potential of approximately −45 mV to near −80 mV [35,38]. In addition, upregulation of the KCa channel activity in HEL cell lines results in Ca2+ -dependent hyperpolarization and increased thrombin- and thapsigargin-stimulated Ca2+ influx [85]. Further roles for KCa3.1 in platelets and megakaryocytes include regulation of volume [92], migration by enhancing store-operated Ca2+ influx [94] and potentiation of procoagulant activity via K+ efflux [93].

Transient receptor potential ion channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

There are approximately 30 transient receptor potential (TRP) ion channels in the mammalian genome, split into six families: TRPC, TRPV, TRPM, TRPP, TRPML and TRPA (reviewed in [95]). They display variable permeabilities to monovalent and divalent cations and play diverse roles in cellular signaling. When first identified, the TRPC (canonical) channels were widely believed to have crucial roles in receptor-operated Ca2+ influx in non-excitable cells, including in platelets; however, this is now unclear (see below). Human platelets have been proposed to express TRPC1, 3, 4, 5 and 6 and TRPV1 [96–98], but some of the work relies on antibodies that have had their specificity questioned (discussed in [99]). TRPV2 and TRPM4 have also been suggested in a proteomics membrane screen [66]. There are no electrophysiological measurements of TRP channels in mammalian platelets, but a combination of molecular and electrophysiological evidence in murine megakaryocytes suggests the presence of TRPC1, TRPC6, TRPM1, TRPM2 and TRPM7 [100]. Interestingly, TRPM2 and TRPM7 have integral enzymatic activity on their intracellular carboxy-terminal tails [95]. TRPM7 is a ubiquitously expressed constitutively active Mg2+ -inhibited channel required for long-term viability, although whether this results from its proposed role in Mg2+ homeostasis is controversial [101,102]. Of interest to platelets are the reports that TRPM7 surface expression is upregulated by shear stress and TRPM2 is activated by oxidative stress [103]. The role(s) of TRPM channels in platelets remains to be investigated.

P2Y1 receptor activation is clearly coupled to activation of a non-selective cation channel in megakaryocytes [7,53,55,100]. The robust expression of TRPC6, together with the electrophysiological properties of this P2Y receptor-evoked cation channel are in agreement with the earlier proposal by Authi and colleagues that TRPC6 forms a pathway for Ca2+ entry activated downstream of PLC [98]. Candidates for activation of TRPC6 include diacylglycerol [98,104], a decrease in PIP2 [7] and more recently protons [105]. No alteration of platelet Ca2+ responses or functional responses could be detected by one group in platelets from mice deficient in either TRPC1 or TRPC6 [99,104]. However, a brief report from another group found that deletion of TRPC6 in mice led to a small increase in bleeding time and a small reduction in arterial thrombosis [106]. Cation channels activated independently of store depletion, for which TRPC6 is a major candidate, have been suggested to play a greater role at higher concentrations of thrombin [107], thus their relative contribution to platelet activation may vary with the strength or type of stimulus and explain the difference between the two murine studies [104,106].

Store-operated Ca2+ channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Probably the most intensively debated topic over the past three decades within the field of platelet ion channels is the identity and activation mechanism of store-operated Ca2+ entry (SOCE). This pathway is opened in response to a reduction in Ca2+ content of the intracellular dense tubular stores and therefore represents a major route for agonist-evoked Ca2+ influx [108]. The first suggestion that SOCE existed in platelets was based upon the close temporal relationship between Ca2+ influx (using Mn2+ entry as a tracer) and Ca2+ release even when a reduction in temperature slowed the formation of IP3 required for store release [109]. However, as agonists can also activate second messenger-operated Ca2+-permeable channels such as TRPC6 (see above), the definitive evidence for SOCE in the platelet and megakaryocyte came from experiments using agents that deplete the internal stores without an increase in IP3, such as ionomycin and the SERCA inhibitor thapsigargin [110–112]. The principal SOCE pathway occurs through CRAC channels, which display distinct characteristics from other Ca2+ channels [113]. These include an extremely high selectivity for Ca2+ over other cations, a single channel conductance in the femtosiemen range, inactivation by physiological elevations of intracellular Ca2+ such that chelators are normally required to measure CRAC currents, and enhancement of channel activation by low concentrations of 2-APB with higher concentrations leading to channel block. The channel also becomes permeable to monovalent cations in the presence of low concentrations of divalent cations. CRAC channels are activated when Stim1 in the endoplasmic reticulum detects a reduced Ca2+ store content (reviewed in [114,115]). Endogenous CRAC currents are very small (in the order of 1 pA/pF), thus with a membrane capacitance of only 100–200 fF, CRAC currents will be < 0.2 pA in whole-platelet recordings and difficult to resolve [116]. In contrast, the average megakaryocyte has 1000-fold higher capacitance, and clear CRAC currents have been recorded in response to P2Y receptor activation and ionomycin-induced store depletion [53,116,117]. The pore-forming subunit of mammalian CRAC channels was first identified as Orai1 in lymphocytes and cell lines [118–120]. Thus, Orai1 was also suggested to be responsible for SOCE in the platelet based on electrophysiological characteristics of megakaryocyte CRAC currents and detection of Orai1 in cDNA generated from purified populations of both human platelets and individually-selected murine megakaryocytes [116]. Conclusive evidence that Orai1 and thus CRAC channels form the major SOCE pathway in murine platelets was shown independently by two groups using platelets lacking functional Orai1 [121,122]. The exact importance of Orai1 in functional responses measured in vitro varies between the two studies, possibly as a result of different experimental conditions, or the fact that one study completely deleted Orai1 [122] and the other inserted a substitute non-functional form (R93W) [121]. Overall, the studies suggest that Orai1 contributes to ex vivo aggregation responses to a greater extent for collagen than for GPCR agonists, although higher collagen concentrations can overcome the loss of response. Furthermore, Orai1 deficiency reduces thrombus formation on collagen surfaces when GPVI is the main stimulus for platelet activation. In vivo Orai1 deficiency substantially reduces arterial thrombosis, pulmonary thromboembolism and cerebral ischemia [122,123], with only a minor prolongation of bleeding time. Thus, Orai1 blockers are a good candidate for anti-thrombotic therapy. Indeed, a recent study demonstrates how Orai1 antagonists inhibit platelet responses and can be used to reduce brain infarction in a murine model of stroke [124]. A key question is the identity of the Ca2+ influx pathway(s) that compensate for the absence of Orai1 to support relatively normal levels of procoagulant activity (due to phosphatidylserine exposure) when platelets are co-stimulated by thrombin and a GPVI stimulus [125].

Neither TRPC1 nor TRPC6 are required for SOCE in murine platelets [99,104]. An important question is whether the mouse studies translate directly to human platelets, particularly as TRPC channels have been proposed to contribute to SOCE in a number of cells, including platelets [126,127]. To date, four families have been diagnosed with different genetic causes of loss of CRAC channel activation: one leads to non-functional CRAC channels, two to loss of Orai1 expression, and one is due to lack of Stim1 expression [115]. The platelet store-operated Ca2+ entry pathway has only been examined in one patient from these families, who completely lacked thapsigargin-evoked Ca2+ entry, consistent with the conclusion from murine studies that Orai1 is the platelet SOCE pathway [128]. The major clinical manifestations reported in patients with dysfunctional CRAC channel activation result from altered function of immune cells, skeletal muscle and sweat glands. Platelet-related symptoms predicted from studies of Orai1-deficient mice resistance are mainly resistance to thrombosis, as platelet counts are normal and tail bleed time is not prolonged [122]. Indeed, patients with abnormal CRAC channel activation show normal or only slightly increased bleeding times. The thrombocytopenia reported in these patients results from autoimmune responses rather than defects in megakaryopoiesis and thrombopoiesis (reviewed in [115]).

Miscellaneous other channels

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Plasma membrane chloride channels have been directly recorded in patch clamp studies in the platelet where membrane depolarization induces their activation [129,130]. It is unclear whether these channels are activated by cytosolic Ca2+, as suggested in whole-cell recordings, as Ca2+ only increased the number of channels induced by depolarization in excised patches. Platelet chloride conductances have been proposed to contribute to volume regulation and the resting membrane potential [34,91,131]. Another chloride conductance reported in the platelet is the cystic fibrosis transmembrane conductance regulator (CFTR) [132], although earlier studies failed to detect the protein [133,134]. CFTR is unique amongst the ATP-binding family of transporters as it acts as an ion channel when ATP is bound. Platelets are hyperactive in cystic fibrosis, but the role of CFTR in the enhanced responses remains unclear as the only suggested roles for this protein in platelets are NO production and platelet-leukocyte interactions [133,134]. Palytoxin is a potent marine toxin that has been reported to induce a non-selective cation channel in murine megakaryocytes [135]. This results from a modification of the gating mechanism of the Na+/K+-ATPase, converting the exchanger into a non-selective cation ion channel [136,137]. Another marine toxin maitotoxin induces an extremely large Ca2+ influx in rabbit platelets, which results in shape change, phosphoinositide hydrolysis and aggregation [138,139]. Studies in other cell types suggest that this action of maitotoxin results from conversion of the plasma membrane Ca2+ -ATPase into a Ca2+ -permeable non-selective cation channel [140,141].

Conclusions and future directions

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

In summary, while significant recent advances have been made in our knowledge of ion channels in platelets, there still remains a paucity of information regarding their contribution to function. Hopefully, the next few years will continue to unravel the relative roles of different surface and endomembrane channel proteins, together with the importance of any accessory proteins. In other cell types, ion channels are already used therapeutically, thus members of this major class of membrane protein may also prove to be useful in the treatment of platelet-related diseases. Indeed, recent in vivo work in murine models demonstrates that intravenous administration of P2X1, gap junction, kainite, AMPA or Orai1 channel inhibitors reduces arterial thrombosis [23,24,77,124,142], which for Orai1 translated into lower levels of neuronal damage after application of a prothrombotic stimulus to the middle cerebral artery [124].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

I am grateful to a number of colleagues for comments on the manuscript and helpful discussions of various ion channel families during the preparation of the review, including R. Evans, S. Amisten, J. Wright, K. Taylor, R. Fern, S. Vaiyapuri and J. Gibbins. Research in the author's laboratory is funded by the British Heart Foundation (PG/11/56 and PG/11/125).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. P2X1 ligand-gated ion channels: the fastest route for agonist-evoked Ca2+ entry in platelets
  5. Ligand-gated cation channels activated by neurotransmitters other than ATP
  6. Organellar ion channels
  7. Gap junctions and hemichannels
  8. Voltage-gated K+ channels
  9. Ca2+ -activated K+ channels
  10. Transient receptor potential ion channels
  11. Store-operated Ca2+ channels
  12. Miscellaneous other channels
  13. Conclusions and future directions
  14. Acknowledgements
  15. Disclosure of Conflict of Interest
  16. References
  17. Supporting Information

Table S1. Summary of ion channels in platelets and megakaryocytes.

FilenameFormatSizeDescription
JTH_4837_sm_table-s1.docx65KSupporting info item

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