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Keywords:

  • calcium;
  • platelet;
  • store-operated calcium entry;
  • stromal interaction molecule 1;
  • thrombosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Summary.  Agonist-induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca2+ release from intracellular stores and Ca2+ entry through the plasma membrane (PM). Ca2+ store release is a well-established process involving phospholipase (PL)C-mediated production of inositol-1,4,5-trisphosphate (IP3), which in turn releases Ca2+ from the intracellular stores through IP3 receptor channels. In contrast, the mechanisms controlling Ca2+ entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, as in other non-excitable cells, the major way of Ca2+ entry involves the agonist-induced release of cytosolic sequestered Ca2+ followed by Ca2+ influx through the PM, a process referred to as store-operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca2+ sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca2+ entry mechanism is mediated by the direct receptor-operated calcium (ROC) channel, P2X1. Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca2+ entry through the PM. This review summarizes the current knowledge of platelet Ca2+ homeostasis with a focus on the newly identified Ca2+ entry mechanisms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Blood platelets are hematopoietic cells produced by bone marrow megakaryocytes. In the intact vasculature most platelets never undergo firm adhesion; however, upon vessel wall injury they rapidly adhere to the exposed extracellular matrix (ECM), become activated and form a platelet plug, thereby preventing blood loss. On the other hand, in atherosclerotic arteries upon plaque rupture the same process can lead to acute vessel occlusion, resulting in life threatening myocardial infarction or ischemic stroke, two of the leading causes of mortality and disability in industrialized countries [1]. Therefore, the activation of platelets has to be regulated with high fidelity to ensure that they become activated only under appropriate conditions.

Platelets possess various adhesion receptors and sophisticated regulatory machinery in order to adhere in response to a well-defined set of stimuli [2]. Platelet activation is triggered by various agonists, including subendothelial collagens, thromboxane A2 (TxA2) and ADP released from activated platelets, and thrombin generated by the coagulation cascade. Although these agonists act on different platelet receptors and trigger different signaling pathways, all lead to an increase in the intracellular Ca2+ concentration ([Ca2+]i) [3].

Ca2+ is an essential second messenger in virtually all cells, regulating a wide range of fundamental cellular processes [4]. In platelets, the elevation in [Ca2+]i contributes to various steps of cellular activation, such as reorganization of the actin cytoskeleton necessary for shape change [5], degranulation or inside-out activation of integrin αIIbß3, indispensable for platelet aggregation [6]. Elevation in [Ca2+]i can derive from two major sources: the release of compartmentalized Ca2+ and the entry of extracellular Ca2+ through the plasma membrane (PM). Whereas the process leading to Ca2+ release from intracellular stores has been well established for several years, the mechanisms underlying Ca2+ entry remained largely unknown until recently. In addition, the relative importance of the two major Ca2+ sources for platelet activation and thrombus formation in vivo has been obscure. In the current review we summarize previous findings and important recent advances in the understanding of platelet Ca2+ homeostasis, with a special focus on the mechanisms of Ca2+ entry and its significance for platelet function.

Calcium store release in platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Agonist-induced stimulation of different platelet receptors leads to the activation of phospholipase (PL) C isoforms, which hydrolyze phosphoinositide-4,5-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and 1,2-diacyl-glycerol (DAG). IP3 in turn induces the release of Ca2+ from intracellular stores while DAG is involved in Ca2+ entry from the extracellular compartment [4,7] (Fig. 1). In platelets, three subfamilies of the PLC enzymes are expressed; namely PLCβ, PLCγ and PLCδ. The predominant isoforms are PLCβ2/3 (in mouse platelets β1 and β3) [8] and PLCγ2 [9]. PLCβ isoforms are solely regulated by G protein-coupled receptors (GPCRs) through the α-subunit of Gq, whereas PLCγ2 is located downstream of the major platelet collagen receptor, glycoprotein (GP) VI [10], the newly identified platelet activating receptor C-type lectin-like receptor 2 (CLEC-2) [11], the adhesion receptor integrins α2β1 and αIIbβ3 [12], the vWF-receptor GPIb-V-IX complex [13] and in human platelets the Fcγ-receptor IIA (FcγRIIA) [14] (Fig. 1). This latter PLC isoform is also regulated by phosphatydilinositol 3-kinase (PI3K) downstream of Gi [15].

image

Figure 1.  The platelet calcium toolkit. Upon receptor activation different phospholipase (PL)C isoforms hydrolyze phosphatidilinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and diacyl-glycerol (DAG). IP3 releases Ca2+ from the intracellular stores and in turn STIM1 opens Orai1 channels in the plasma membrane, a process called store-operated calcium entry (SOCE), whereas DAG mediates non-SOCE through canonical transient receptor potential channel 6 (TRPC6). Additionally, a direct receptor-operated calcium (ROC) channel, P2X1, and a Na+/ Ca2+ exchanger (NCX) contribute to the elevation in [Ca2+]i. The counteracting mechanisms involve sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs) and plasma membrane Ca2+ ATPases (PMCAs), which pump Ca2+ back into the stores or through the plasma membrane out of the cell, respectively. IP3R, IP3-receptor; ATP, adenosine triphosphate; ADP, adenosine diphosphate; GPVI, glycoprotein VI; FcRγ, Fc receptor γ chain; FcγRIIa, Fc γ receptor IIa; CLEC-2, C-type lectin-like receptor 2; PI3-K, phosphatidylinositol 3-kinase; Syk, spleen tyrosine kinase. Due to controversies about the localization and role of TRPC1 in the literature, this protein is not depicted in the figure.

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IP3 generated by PLCs releases Ca2+ from the intracellular stores by directly activating IP3 receptors (IP3-R) in the ER, which themselves are Ca permeable ion channels (Fig. 1). Additionally, the expression of IP3-Rs in the PM has been reported in a single study and suggested to be involved in cation entry [16]. Three different isoforms of IP3-Rs have been cloned in mammalian cells, the relative IP3-binding affinity of which is as follows: IP3-R2 > IP3-R1 > IP3-R3 [17]. In platelets all three isoforms have been found; however, the predominant ones are IP3-R1 and IP3-R2. The regulation of these receptors is supposed to occur through receptor phosphorylation, most probably by different protein kinases (PKs). A number of studies reported inhibition of receptor function after phosphorylation [18–20]; however, activatory effect of phosphorylation has also been proposed [21]. The reason for these differences is unknown and requires further investigation. The inhibitory effect of phosphorylation seems to be mediated by cyclic nucleotides (cAMP and cGMP) [19,20,22,23].

Major calcium stores in platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Although there are several intracellular organelles that contain higher [Ca2+] than the surrounding cytoplasm and could therefore act as possible releasable Ca2+ stores, there is consensus that the major intracellular Ca2+ pool in most mammalian cells is the endoplasmic reticulum (ER). In platelets, the existence of two distinct Ca2+ stores has been proposed based on the expression of two different sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms [24–26], and on their sensitivity towards the SERCA inhibitor thapsigargin. A 100 kDa SERCA isoform (identified as SERCA2b [27]) showing high sensitivity to thapsigargin is expressed in one of the proposed stores that corresponds to the dense tubular system (DTS), the equivalent of the ER in platelets. The 97 kDa SERCA isoform (SERCA3 [28,29]) is expressed in an acidic organelle that shows low sensitivity to thapsigargin but is sensitive to 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ) [25]. The true identity of this store, however, remains to be clarified [30] (for review see reference [31]).

Store-operated calcium entry in platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

In non-excitable cells, such as platelets, IP3 mediated Ca2+ release from the intracellular stores triggers Ca2+ entry from the extracellular compartment. This mechanism, although at that time believed to be Ca triggered, was first described in neutrophils in 1986 [32]. Since then it became clear that the Ca entry is store mediated [33] and it is now referred to as store-operated calcium entry (SOCE) [34]. Whereas this process has been described in many cell types for over two decades and several hypotheses evolved on the nature of the molecular link between Ca2+ store release and SOCE [35], the underlying mechanisms have remained elusive. In platelets, conformational coupling between the IP3-R2 and a store-operated calcium (SOC) channel candidate, canonical transient receptor potential (TRPC) channel 1, was proposed [36,37] (see below). Despite the efforts, neither the identification of the exact mechanism or the key proteins succeeded, nor could the relevance of SOCE for cell physiology be elucidated, until recently.

STIM1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

The breakthrough in our understanding of the process was the identification of stromal interaction molecule 1 (STIM1), described as the Ca sensor molecule in the ER. In 2005, two independent groups identified STIM1 – based on RNA interference (RNAi)-based screening and knock-down – as a conserved component of SOCE in drosophila S2 cells, HeLa cells and Jurkat T cell lines [38–40].

STIM1 is a type I single transmembrane protein containing two N-terminal EF hand domains (a canonical and a hidden one), followed by a sterile α-motif (SAM) domain, the transmembrane region, two coiled-coil region and at the C-terminal end a serine/proline-rich and a lysine-rich domain [41,42] (Fig. 2). The EF hand domains of the molecule are situated in the ER lumen and bind Ca2+. Upon store release, this Ca2+ binding is disturbed, and STIM1 redistributes to puncta and opens the SOC channels in the plasma membrane. One critical experiment to demonstrate this was the introduction of a point mutation in the ‘canonical’ EF hand of STIM1. This results in impaired Ca2+ binding thereby mimicking store-release and activating STIM1. As a consequence, EF hand mutant STIM1 keeps the SOC channels permanently opened, leading to continuous Ca2+ influx from the extracellular space as shown by in vitro studies [38,40,43] (Fig. 3).

image

Figure 2.  Protein structure of STIM1. STIM1 is a type I single transmembrane protein in the endoplasmic reticulum (ER) membrane. It bears two N-terminal EF hand domains (a canonical and a ‘hidden’ one) located in the ER lumen, which play a central role in the Ca2+ binding of the protein. SAM, sterile α-motif; TM, transmembrane domain; CC, coiled-coil domain.

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image

Figure 3. Store-operated calcium entry (SOCE) in platelets. In platelets, distinct subtypes of surface receptors activate two major isoforms of PLC; G-protein coupled receptors (GPCRs) activate PLCβ through Gq, whereas integrins and receptors coupled to an immunoreceptor tyrosine-based activation motif (ITAM) activate PLCγ2 through Syk. Receptor agonist binding results in calcium store release through inositol-1,4,5-trisphosphate receptors (IP3-R) in the endoplasmic reticulum (ER) membrane. This disrupts the calcium binding of the EF hand domain of STIM1 in the ER lumen and leads to the activation and redistribution of STIM1 to plasma membrane (PM) near puncta where it opens Orai1, the major store-operated calcium (SOC) channel in the PM, to allow calcium entry.

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The first description of an in vivo function of STIM1 came from platelets[43] of mice bearing a single amino acid change (D84G) in the canonical EF hand of the molecule (Sax; named after the Bulgarian king Saxcoburgotski who suffered from a bleeding disorder). Animals homozygous for the mutation (Sax/Sax) died in utero due to severe hemorrhages in different regions of the body, whereas heterozygous mice (Sax/+) were born according to the Mendelian ratio, still displaying multiple defects. Ca2+ measurements in Stim1Sax/+ platelets showed elevated basal Ca2+ levels compared with wild-type controls. This turned out to be the result of permanently opened SOC channels in the plasma membrane, providing in vivo evidence that an EF hand mutation in STIM1 results in gain-of-function. Platelets were found to be in a preactivated state and were rapidly cleared from the circulation, manifesting in macrothrombocytopenia and a bleeding phenotype. Interestingly, Stim1Sax/+ platelets displayed a selective defect in PLCγ2-signaling, as evidenced by impaired αIIbβ3 activation and degranulation, whereas these processes were only mildly affected in response to PLCβ-stimulation. These observations indicated an important function of STIM1 in platelets and suggested it to be the long-sought Ca2+ sensor in these cells linking store-release and Ca2+ entry. Definitive proof for this came shortly after from studies on STIM1 knock-out mice. Parallel to the description of defective T cell and mast cell function in the absence of STIM1 [44,45], STIM1-mediated SOCE was found to be essential also for platelet function [46]. Intracellular Ca2+ measurements showed an almost complete lack of SOCE in Stim1−/− platelets (Fig. 4) and severely impaired Ca2+ responses to all major platelet agonists. It is noteworthy that in accordance with the results on mast cells, reduced Ca2+ release from internal stores upon agonist-induced platelet activation was observed. This probably reflects a lower filling state of the ER because similar results were obtained after passively emptying the stores with thapsigargin (Fig. 4). Thus, STIM1 is essential for SOCE in platelets, but is also involved in the regulation of store [Ca2+] by yet-to-be defined mechanisms.

image

Figure 4.  Functional SOCE is essential for stable thrombus formation in vivo. (A) Store release and SOCE was studied in platelets of the indicated mouse lines using Fura-2 loaded platelets and fluorimetric intracellular calcium measurements. The store content was estimated by blocking the SERCA pumps in the ER using thapsigargin, whereas SOCE was measured by the subsequent addition of extracellular calcium. Representative curves and mean changes in the intracellular calcium concentration (Δ[Ca2+]i) plus or minus SD. (B) Thrombus formation in Stim1−/− and Orai1−/− bone marrow chimeras was studied in a FeCl3-induced chemical injury model of small mesenteric arteries (upper line) and in a mechanical injury model of the abdominal aorta (middle line). Impaired thrombus formation was observed with both mouse strains in the aorta model, whereas only Stim1−/− mice showed defects in the chemical injury model.

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Despite the dramatically reduced Ca2+ responses to both PLCβ and γ activating platelet agonists, Stim1−/− platelets showed unaltered αIIbβ3 activation, aggregation and degranulation through the GPCR/PLCβ pathway and only mild defects in response to GPVI/PLCγ2 agonists in the absence of flow in vitro. An explanation for this could be that ∼10% Ca2+ entry still occurred in these platelets (Fig. 4), which might be sufficient to support those functions. The residual Ca2+ entry was possibly non-SOCE mediated by TRPC6 or P2X1 (see below). This rather mild activation deficit in the mutant platelets turned into a dramatically reduced capability to form stable three-dimensional aggregates on collagen under high shear conditions in a whole blood perfusion assay ex vivo. These experimental conditions mimic important aspects of thrombus formation in flowing blood in vivo where locally produced soluble mediators are rapidly cleared. Thus, STIM1-dependent SOCE appears to be of particular importance for platelet function under flow conditions; however, the underlying mechanisms are not clear. It is also not clear why defective SOCE has a stronger impact on platelet responses to GPVI agonists compared with Gq-coupled agonists as seen in both Stim1Sax/+ and Stim1−/− mice [43,46], but it could be related to the fact that GPVI and GPCRs activate different PLC isoforms. The low [Ca2+]i alone cannot explain the cellular activation defect downstream of GPVI/PLCγ2 as similar or even lower levels are seen in response to thrombin and ADP, respectively, without causing this defect. Possibly, STIM1 plays a role in GPVI/PLCγ2 signaling independently of Ca2+ regulation, but more experiments are needed to test this hypothesis.

Orai1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Parallel to STIM1, calcium-release activated calcium modulator 1 (CRACM1 or Orai1), a plasma membrane protein with four predicted transmembrane domains and intracellular C- and N-termini, was identified as another essential component of SOCE by the analysis of T cells from patients with severe combined immunodeficiency (SCID) and in Drosophila cells [47,48]. Shortly after, Orai1 was shown to be the pore forming unit of the SOC channel [49,50] and it was also shown that Orai1 multimerizes with itself to create the channel [51]. In vivo studies of mast cell [52], T and B cell function [53] in Orai1 knock-out mice confirmed the in vitro findings. Tolhurst and coworkers showed by quantitative RT-PCR analysis of primary murine megakaryocytes, human platelets and megakaryocytic cell lines that there were high expression levels of Orai1 in all three cases, well above that of TRPC1 or other TRPCs [54]. Shortly later this was confirmed independently and the analysis of Orai1−/− platelets unambiguously established Orai1 as the major SOC channel on the platelet surface [55,56]. Using RT-PCR, human and mouse platelets were shown to express all three isoforms of the Orai channel family (Orai1-3), with Orai1 being the predominant isoform in both species, and Western-blot analysis of human platelet lysates detected strong expression of the Orai1 protein. Intracellular Ca2+ measurements on Orai1−/− platelets showed ∼90% reduction of SOCE (Fig. 4), which correlates well with the results from Stim1−/− platelets [46]. However, in contrast to Stim1−/− platelets, Orai1−/− platelets displayed normal Ca2+ store content and, consequently, agonist-induced Ca2+ store release (Fig. 4). Therefore, Orai1−/− platelets reach consistently higher [Ca2+]i than Stim1−/− platelets in response to all tested agonists and this difference may be relevant for thrombus stability in vivo under certain (experimental) conditions, such as FeCl3-induced thrombosis [55] (Fig. 4). Furthermore, these data suggest that STIM1 contributes to the regulation of the Ca2+ store content in platelets independently of Orai1 (i.e. SOCE). Studies of platelet function in Orai1−/− mice revealed a phenotype very similar to that observed in Stim1−/− mice, with only minor defects in GPVI signaling observed in the absence of flow, but markedly reduced aggregate stability under flow conditions.

The function of Orai1 as the major SOC channel in platelets has also recently been demonstrated in an independent study utilizing a R93W Orai1 mutant mouse line [57]. This mutation is modeled on the mutation observed in patients with severe combined immunodeficiency (SCID). Although similar in its conclusion (e.g. Orai1 is the major SOC channel in platelets) there are some differences between the results of this study and that of the Orai1−/− study. Whereas under static conditions Orai1−/− platelets show a rather mild and selective GPVI signaling defect, Orai1R93W platelets show an integrin activation defect both through GPVI and GPCRs without major differences in aggregation and thrombus formation under flow. One possible explanation of this is the use of slightly different experimental conditions (different agonist concentration and shear forces). Alternatively, as also mentioned by Bergmeier et al. [57], the R93W mutation may not completely abolish Orai1 function.

Taken together, these recent advances have now established STIM1 and Orai1 as the key players of SOCE in platelets, with STIM1 being responsible for the detection of Ca2+ release from the intracellular stores and for regulation of Orai1, the major SOC channel on the platelet surface (Fig. 3).

One surprising finding of the above studies is that platelets of Stim1−/− and Orai1−/− mice, despite a virtual complete lack of agonist-induced Ca2+ entry, are still able to fulfill most of their physiological functions. Thus, Ca2+ store-release appears to be sufficient to trigger most responses such as shape change, integrin activation and granule release directly. Considering that platelets, unlike most other non-excitable cells, have to respond very fast to activating stimuli, it is plausible that basic cellular functions must be regulated without the delay caused by an indirect process such as SOCE, which involves molecular coupling that requires up to 15 s to be accomplished [58]. Thus, SOCE may be of particular relevance for processes following initial adhesion, primary activation and aggregation, such as thrombus stabilization, clot retraction, coagulant activity or possibly recruitment of other cells to the site of injury. In line with this notion, a major observation made in Stim1−/− and Orai1−/− mice is reduced thrombus stability in vivo (see below) and reduced PS exposure of Orai1R93W platelets [57].

TRPC1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Members of the transient receptor potential (TRP) channel family have also been suggested as candidate molecules to fulfill the function of the major SOC channel in platelets. The TRP family can be further divided into seven subfamilies, of which the canonical TRPs (TRPCs) are the best described. Platelets have been shown to express members of the TRPC but also of the TRPM subfamily [59–61]. As mentioned above, the conformational coupling model proposed TRPC1 to be a major SOC channel in platelets based mainly on in vitro studies, where a blocking antibody against TRPC1 reduced SOCE in response to thrombin and thapsigargin [62]. The model suggested that Ca2+ store release triggers a de novo coupling between the IP3-R2 and TRPC1, thereby activating TRPC1 as SOC channel in the plasma membrane to allow Ca2+ entry [36,63]. There are, however, numerous controversies in the literature regarding the TRPC channel family and its role in platelet function. While TRPC1 has been reported to be expressed in lipid rafts of the platelet plasma membrane associated with TRPC4 and TRPC5 [64], others found it to be expressed only in internal membranes [65] and in barely detectable amounts [65,66]. Furthermore, there are reports stating no effect of anti-TRPC1 treatment on thrombin- or thapsigargin-induced SOCE [59,66].

Studies on TRPC1 knock-out mice found no significant differences in Ca2+ homeostasis or function of TRPC1−/− platelets [66] (Fig. 4), also arguing against a major role of the protein for SOCE in platelets. This was further confirmed by experiments where TRPC1−/− mice were crossed with the EF hand mutant Stim1Sax/+ mice, in which platelet SOC channels are constitutively opened [43]. In this setting, lack of TRPC1 did not rescue the macrothrombocytopenic phenotype of the Stim1Sax/+ mice and the basal Ca2+ level in their platelets remained elevated [66]. If TRPC1 was the major SOC channel on the platelet surface, lack of the protein should have at least ameliorated these phenotypes.

One explanation for the different data from previous reports suggesting TRPC1 to be the major SOC channel on the platelet surface could be the use of antibodies of questionable selectivity towards the protein [66,67]. A compensatory effect of other TRPCs in the TRPC1−/− mice is unlikely because TRPC6 is the only member of the TRPC channel family shown to be expressed in murine platelets and expression levels of the protein were unaltered. Thus, further studies will be required to identify the exact role of TRPC1 in platelets.

TRPC6

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Besides SOCE, other Ca2+ entry mechanisms also exist in platelets. These are the so-called non-store-operated calcium entry (non-SOCE) mediated by DAG and a direct, receptor operated calcium entry (ROCE) through the purinergic receptor, P2X1 (Fig. 1). As discussed above, PLC isoforms hydrolyze PIP2 to IP3 and DAG. Whilst IP3 is responsible for Ca2+ release from the intracellular stores, DAG is known to directly activate Ca2+ channels in the plasma membrane, thereby facilitating non-SOCE [4]. This mechanism exists also in platelets and TRPC6 has been demonstrated to form a non-SOC channel [65] (Fig. 1). Unlike in the case of TRPC1, there is consensus about the robust and functional expression of TRPC6 in both human and murine megakaryocytes/platelets [60,61,64,65]. Hassock et al. proposed TRPC6 as the non-SOC channel in platelets, based on data showing TRPC6 activation downstream of PLC but independent of Ca2+ release from the intracellular stores. They further showed that TRPC6 becomes phosphorylated by PKA and PKG; however, this phosphorylation does not affect Ca2+ entry through the channel [65].

Jardin et al. in contrast suggest that TRPC6 is not only involved in non-SOCE, but also in SOCE in platelets [68]. In studies where platelets were incubated with an anti-TRPC6 antibody, they found that besides non-SOCE mediated by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), SOCE evoked by thrombin, thapsigargin or TBHQ was also reduced. The reason for the discrepancies between the two studies is unknown and further investigation of the role of TRPC6 in platelet physiology is required.

P2X1

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

The presence of a direct receptor-operated calcium channel on the platelet surface was first reported in the early 1990s [69] and was proposed to be the P2X1 purinoceptor [70]. A few years later, Vial et al. confirmed these results and identified the channel unambiguously as the P2X1 receptor (Fig. 1), one of seven distinct P2X receptors cloned from mammalian tissues [71]. The natural agonist of the platelet P2X1 receptor is ATP, whereas ADP has been reported in a single study to have an inhibitory effect [72]. Fast receptor desensitization through the release of purines during platelet handling and the slow recovery of the receptor made it difficult to investigate the functional importance of P2X1. Despite these technical problems, it became clear from in vitro studies – using high concentrations of apyrase to scavenge the released ADP – that platelet activation through the P2X1 receptor can induce reversible shape change [73] and through the PKC/Erk2 pathway contributes to complete dense granule secretion [74]. This contribution occurs through the centralization of granules without release of granule content [74]. The significance of these P2X1 mediated platelet responses seems to be the amplification of signals mediated by GPCR agonists or collagen, especially at low agonist concentrations [75–77]. It is also likely that Ca2+ influx through P2X1 and the consequential membrane depolarization activates PLCs and IP3-receptors, thereby providing further enhancement of the Gq/PLCβ and GPVI/PLCγ2 mediated activation processes [78,79].

The generation of P2X1 transgenic mice provided definitive evidence for an important role of this ROC channel in platelet function [80,81]. P2X1−/− platelets aggregated less effectively and showed decreased granule release in response to low doses of the GPVI agonist collagen, whereas they responded normally to GPCR agonists and higher doses of collagen. Under flow conditions, platelets from P2X1−/− blood showed reduced thrombus forming capacity at higher arterial shear rates [80]. Conversely, overexpression of the receptor in mice resulted in a prothrombotic phenotype [81].

Membrane Ca2+ ATPases in platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

As already very small increases in [Ca2+]i lead to platelet activation [43,73], the maintenance of a stable [Ca2+]i is essential to keep platelets in a resting state in the circulation, thereby preventing undesired thrombus formation. However, there is continuous leakage of Ca2+ from the intracellular stores, which in the absence of counteracting mechanisms would rapidly lead to platelet activation. Two distinct counteracting mechanisms have been identified in platelets: sarcoplasmic/endoplasmic Ca2+ ATPases (SERCAs) pump Ca2+ back to the intracellular stores, whereas plasma membrane Ca2+ ATPases (PMCAs) pump it out of the cell [82] (Fig. 1).

Platelets are known to express two SERCA isoforms (SERCA2b and 3) and two PMCA isoforms (PMCA1b and 4b) [25,27–29,83,84] but not much is known about their regulation and relevance for platelet function. PMCA activity in different tissues has been shown to be regulated by Ca2+/calmodulin, PKA and PKC, acidic phospholipids and proteolysis (reviewed in Ref. [85]). In platelets, an inhibitory role for tyrosine phosphorylation at position 1176 of PMCA4b has been proposed [83], and enhanced phosphorylation of PMCA isoforms seems to be responsible for the observed elevation in [Ca2+]i in platelets from hypertensive patients[86]. A further study found that small GTPases of the Ras family are involved in this phosphorylation process [87]. In the case of platelet SERCA isoforms, SERCA3b has been reported to be regulated by Ras-proximate (Rap) 1b [88]. In this paper the authors report decreased SERCA3b activity as a consequence of decreased phosphorylation of Rap1b in spontaneously hypertensive rats. Finally, both SERCA and PMCA activity has been suggested to be modulated by reactive oxygen species [89].

There are only a few studies that address the physiological relevance of these two Ca2+ removal mechanisms in resting or activated platelets [87,90]. They came to the conclusion that in resting platelets the extrusion of Ca2+ through PMCAs is the main way of regulation [87], whereas after platelet stimulation the activation of SERCA pumps precedes that of PMCA pumps, although the action of PMCAs lasts longer [90]. Because of the suicidal character of platelets, upon activation they may not require fast Ca2+ export or storage conducted by PMCAs or SERCAs, respectively. Therefore, the above described results on the regulation of Ca2+ ATPases in platelets in vitro require in vivo confirmation. Several mouse lines lacking individual SERCA/PMCA isoforms have been reported in recent years (for review see Ref. [91]). However, to date platelet function has not been assessed in these animals.

An additional way of Ca2+ removal from the platelet cytoplasm has been reported in the early 1990s [92]. This is a Na+/ Ca2+ exchanger (NCX), which is able to accomplish very fast Ca2+ extrusion through the plasma membrane and can also work in a reverse mode after Na+ loading of the cytoplasm. Recently, Ca2+ entry through the NCX was suggested to play an important role in SOCE as a consequence of Na+ entry through the SOC channel [93] (Fig. 1).

Platelet calcium entry mechanisms as potential therapeutic targets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

Ca2+ is a central and common second messenger downstream of most signaling pathways in platelets. Therefore, regulators of Ca2+ signaling might be interesting targets for platelet inhibition [56]. Indeed, in vivo studies on mice lacking molecules involved in agonist-induced Ca2+ entry have confirmed this notion in that strong antithrombotic protection but no or only a mild hemostatic defect was seen in those animals. The platelet ROC channel, P2X1, is one of the candidate molecules. Mice deficient in the protein were found to be protected in models of collagen/epinephrine-induced pulmonary thromboembolism and localized, laser-induced arterial thrombosis, whereas bleeding time was normal in most of the cases. Similarly, mice lacking major components of the SOCE machinery, namely STIM1 or Orai1, showed significant protection in different in vivo thrombosis models due to reduced thrombus stability, but only mildly prolonged bleeding times (Fig. 4). Furthermore, in a model system of ischemic stroke, where infarct development is known to be dependent on platelet function [94], both Stim1−/− and Orai1−/− bone marrow chimeras were found to be largely protected against ischemia/reperfusion induced brain injury without any detectable tendency towards intracerebral hemorrhage [46,55] (Fig. 4). Importantly, this protection was clinically relevant, because the global neurological and motoric functions of these animals were significantly better 24 h after ischemia than those of control mice. One major limitation of the use of STIM1 or Orai1 as antithrombotic targets might be, however, that both are widely expressed molecules and that, as shown in the case of the Orai1 R93W mutation which results in SCID, mutations or lack of these molecules result in severe immunological deficits. Therefore cell-specific targeting of platelets is required.

Still, these in vivo studies have revealed that the different Ca2+ entry mechanisms are of particular importance for pathological thrombus formation without having a major impact on hemostasis. This makes molecules regulating Ca2+ entry in platelets a promising therapeutic target for the prevention and treatment of ischemic cardio- and cerebrovascular events.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

During the past 5 years, significant progress has been made in understanding platelet Ca2+ homeostasis in general and Ca2+ entry pathways in particular. The mechanisms regulating Ca2+ release from intracellular stores have been well known for some time and now there is an increasing body of knowledge regarding the different ways of Ca2+ influx through the plasma membrane. Using the advantages of knock-out mouse models, many previous in vitro findings could be confirmed, whilst some others turned out to be false. The purinergic receptor P2X1 has been shown to be indeed responsible for very fast receptor-operated Ca2+ entry in platelets, whereas in the case of SOCE a key role of STIM1 and Orai1 emerged. Not surprisingly, these recent advances not only answered questions but also raised new ones. For example, it will be important to clarify whether the interaction between STIM1 and Orai1 is exclusive, or STIM1 also regulates other channel proteins in platelets. Other obvious questions remain. How is STIM1-Orai1 interaction regulated? Why do the two major signaling pathways in platelets show differences in their dependency on SOCE? What role do the different PMCA and SERCA isoforms play in platelets in vivo? Answering these and other questions will further help to understand platelet function in its complexity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References

We would like to thank all members of our laboratory for the help with the work on platelet Ca2+ homeostasis and for the very useful suggestions and critical discussion during the preparation of this review. We also thank S. P. Watson for critically reading the manuscript and for his helpful suggestions. Our work on Ca2+ regulation in platelets was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereiche 688 and 487) and the Rudolf Virchow Center.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Calcium store release in platelets
  5. Major calcium stores in platelets
  6. Store-operated calcium entry in platelets
  7. STIM1
  8. Orai1
  9. TRPC1
  10. TRPC6
  11. P2X1
  12. Membrane Ca2+ ATPases in platelets
  13. Platelet calcium entry mechanisms as potential therapeutic targets
  14. Conclusion
  15. Acknowledgements
  16. Disclosure of Conflict of Interests
  17. References
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