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Journal of Thrombosis and Haemostasis, 7: 1057–1066 DOI: 10.1111/j.1538-7836.2009.03455.

REVIEW ARTICLE

Calcium signaling in platelets


D . V A R G A - S Z A B O , *   A . B R A U N * and B . N I E S W A N D T *
*Chair of Vascular Medicine and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Würzburg;
and  Surgical Department, Helios Klinikum Wuppertal, Wuppertal, Germany

To cite this article: Varga-Szabo D, Braun A, Nieswandt B. Calcium signaling in platelets. J Thromb Haemost 2009; 7: 1057–66.

to acute vessel occlusion, resulting in life threatening myocar-


Summary. Agonist-induced elevation in cytosolic Ca2+ con- dial infarction or ischemic stroke, two of the leading causes of
centrations is essential for platelet activation in hemostasis and mortality and disability in industrialized countries [1]. There-
thrombosis. It occurs through Ca2+ release from intracellular fore, the activation of platelets has to be regulated with high
stores and Ca2+ entry through the plasma membrane (PM). fidelity to ensure that they become activated only under
Ca2+ store release is a well-established process involving appropriate conditions.
phospholipase (PL)C-mediated production of inositol-1,4,5- Platelets possess various adhesion receptors and sophisti-
trisphosphate (IP3), which in turn releases Ca2+ from the cated regulatory machinery in order to adhere in response to a
intracellular stores through IP3 receptor channels. In contrast, well-defined set of stimuli [2]. Platelet activation is triggered by
the mechanisms controlling Ca2+ entry and the significance of various agonists, including subendothelial collagens, throm-
this process for platelet activation have been elucidated only boxane A2 (TxA2) and ADP released from activated platelets,
very recently. In platelets, as in other non-excitable cells, the and thrombin generated by the coagulation cascade. Although
major way of Ca2+ entry involves the agonist-induced release of these agonists act on different platelet receptors and trigger
cytosolic sequestered Ca2+ followed by Ca2+ influx through different signaling pathways, all lead to an increase in the
the PM, a process referred to as store-operated calcium entry intracellular Ca2+ concentration ([Ca2+]i) [3].
(SOCE). It is now clear that stromal interaction molecule 1 Ca2+ is an essential second messenger in virtually all cells,
(STIM1), a Ca2+ sensor molecule in intracellular stores, and the regulating a wide range of fundamental cellular processes [4]. In
four transmembrane channel protein Orai1 are the key players platelets, the elevation in [Ca2+]i contributes to various steps of
in platelet SOCE. The other major Ca2+ entry mechanism is cellular activation, such as reorganization of the actin cyto-
mediated by the direct receptor-operated calcium (ROC) skeleton necessary for shape change [5], degranulation or
channel, P2X1. Besides these, canonical transient receptor inside-out activation of integrin aIIbß3, indispensable for
potential channel (TRPC) 6 mediates Ca2+ entry through the platelet aggregation [6]. Elevation in [Ca2+]i can derive from
PM. This review summarizes the current knowledge of platelet two major sources: the release of compartmentalized Ca2+ and
Ca2+ homeostasis with a focus on the newly identified Ca2+ the entry of extracellular Ca2+ through the plasma membrane
entry mechanisms. (PM). Whereas the process leading to Ca2+ release from
intracellular stores has been well established for several years,
Keywords: calcium, platelet, store-operated calcium entry, the mechanisms underlying Ca2+ entry remained largely
stromal interaction molecule 1, thrombosis. unknown until recently. In addition, the relative importance
of the two major Ca2+ sources for platelet activation and
Introduction thrombus formation in vivo has been obscure. In the current
review we summarize previous findings and important recent
Blood platelets are hematopoietic cells produced by bone
advances in the understanding of platelet Ca2+ homeostasis,
marrow megakaryocytes. In the intact vasculature most
with a special focus on the mechanisms of Ca2+ entry and its
platelets never undergo firm adhesion; however, upon vessel
significance for platelet function.
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 athero- Calcium store release in platelets
sclerotic arteries upon plaque rupture the same process can lead
Agonist-induced stimulation of different platelet receptors
Correspondence: Bernhard Nieswandt, Rudolf Virchow Center, DFG
leads to the activation of phospholipase (PL) C isoforms, which
Research Center for Experimental Biomedicine, University of hydrolyze phosphoinositide-4,5-bisphosphate (PIP2) to inosi-
Würzburg, Zinklesweg 10, 97078 Würzburg, Germany. tol-1,4,5-trisphosphate (IP3) and 1,2-diacyl-glycerol (DAG).
Tel.: +49 931 201 44060; fax: +49 931 201 44068. IP3 in turn induces the release of Ca2+ from intracellular stores
E-mail: bernhard.nieswandt@virchow.uni-wuerzburg.de while DAG is involved in Ca2+ entry from the extracellular

Ó 2009 International Society on Thrombosis and Haemostasis


1058 D. Varga-Szabo et al

compartment [4,7] (Fig. 1). In platelets, three subfamilies of the phorylation [18–20]; however, activatory effect of phosphory-
PLC enzymes are expressed; namely PLCb, PLCc and PLCd. lation has also been proposed [21]. The reason for these
The predominant isoforms are PLCb2/3 (in mouse platelets b1 differences is unknown and requires further investigation. The
and b3) [8] and PLCc2 [9]. PLCb isoforms are solely regulated inhibitory effect of phosphorylation seems to be mediated by
by G protein-coupled receptors (GPCRs) through the a- cyclic nucleotides (cAMP and cGMP) [19,20,22,23].
subunit of Gq, whereas PLCc2 is located downstream of the
major platelet collagen receptor, glycoprotein (GP) VI [10], the
Major calcium stores in platelets
newly identified platelet activating receptor C-type lectin-like
receptor 2 (CLEC-2) [11], the adhesion receptor integrins a2b1 Although there are several intracellular organelles that contain
and aIIbb3 [12], the vWF-receptor GPIb-V-IX complex [13] higher [Ca2+] than the surrounding cytoplasm and could
and in human platelets the Fcc-receptor IIA (FccRIIA) [14] therefore act as possible releasable Ca2+ stores, there is
(Fig. 1). This latter PLC isoform is also regulated by consensus that the major intracellular Ca2+ pool in most
phosphatydilinositol 3-kinase (PI3K) downstream of Gi [15]. mammalian cells is the endoplasmic reticulum (ER). In
IP3 generated by PLCs releases Ca2+ from the intracellular platelets, the existence of two distinct Ca2+ stores has been
stores by directly activating IP3 receptors (IP3-R) in the ER, proposed based on the expression of two different sarcoplas-
which themselves are Ca2± permeable ion channels (Fig. 1). mic/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms
Additionally, the expression of IP3-Rs in the PM has been [24–26], and on their sensitivity towards the SERCA inhibitor
reported in a single study and suggested to be involved in cation thapsigargin. A 100 kDa SERCA isoform (identified as
entry [16]. Three different isoforms of IP3-Rs have been cloned SERCA2b [27]) showing high sensitivity to thapsigargin is
in mammalian cells, the relative IP3-binding affinity of which is expressed in one of the proposed stores that corresponds to the
as follows: IP3-R2 > IP3-R1 > IP3-R3 [17]. In platelets all dense tubular system (DTS), the equivalent of the ER in
three isoforms have been found; however, the predominant platelets. The 97 kDa SERCA isoform (SERCA3 [28,29]) is
ones are IP3-R1 and IP3-R2. The regulation of these receptors expressed in an acidic organelle that shows low sensitivity to
is supposed to occur through receptor phosphorylation, most thapsigargin but is sensitive to 2,5-di-(t-butyl)-1,4-hydro-
probably by different protein kinases (PKs). A number of quinone (TBHQ) [25]. The true identity of this store, however,
studies reported inhibition of receptor function after phos- remains to be clarified [30] (for review see reference [31]).

ADP, TxA2 Ca2+


thrombin
collagen CLEC-2
TRPC6
GPVI PLCβ Gq
PIP2 DAG
Y
FcRy X Ca2+
X
Ca2+
k

L
ai1
Sy

FcyRlla Ca2+ Or
PLCγ2
integrin STIM1
Sy Ca2+
Ca2+
β k IP3
α Ca2+ Ca2+ Ca
2+
IP3R
Ca2+
SERCA
SERCA

Pl3-K Ca2+
Gi Ca2+
Ca2+ Or
ai1
Ca2+
X1
ATP P2 Ca2+
PMCA
PMCA

Ca2+
NCX

Ca2+

Fig. 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; FcRc, Fc receptor c chain; FccRIIa, Fc c 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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Calcium signaling in platelets 1059

situated in the ER lumen and bind Ca2+. Upon store release,


Store-operated calcium entry in platelets
this Ca2+ binding is disturbed, and STIM1 redistributes to
In non-excitable cells, such as platelets, IP3 mediated Ca2+ puncta and opens the SOC channels in the plasma membrane.
release from the intracellular stores triggers Ca2+ entry from One critical experiment to demonstrate this was the introduc-
the extracellular compartment. This mechanism, although at tion of a point mutation in the ÔcanonicalÕ EF hand of STIM1.
that time believed to be Ca2± triggered, was first described in This results in impaired Ca2+ binding thereby mimicking store-
neutrophils in 1986 [32]. Since then it became clear that the release and activating STIM1. As a consequence, EF hand
Ca2± entry is store mediated [33] and it is now referred to as mutant STIM1 keeps the SOC channels permanently opened,
store-operated calcium entry (SOCE) [34]. Whereas this process leading to continuous Ca2+ influx from the extracellular space
has been described in many cell types for over two decades and as shown by in vitro studies [38,40,43] (Fig. 3).
several hypotheses evolved on the nature of the molecular link The first description of an in vivo function of STIM1 came
between Ca2+ store release and SOCE [35], the underlying from platelets[43] of mice bearing a single amino acid change
mechanisms have remained elusive. In platelets, conforma- (D84G) in the canonical EF hand of the molecule (Sax; named
tional coupling between the IP3-R2 and a store-operated after the Bulgarian king Saxcoburgotski who suffered from a
calcium (SOC) channel candidate, canonical transient receptor bleeding disorder). Animals homozygous for the mutation
potential (TRPC) channel 1, was proposed [36,37] (see below). (Sax/Sax) died in utero due to severe hemorrhages in different
Despite the efforts, neither the identification of the exact regions of the body, whereas heterozygous mice (Sax/+) were
mechanism or the key proteins succeeded, nor could the born according to the Mendelian ratio, still displaying multiple
relevance of SOCE for cell physiology be elucidated, until defects. Ca2+ measurements in Stim1Sax/+ platelets showed
recently. 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
STIM1
that an EF hand mutation in STIM1 results in gain-of-
The breakthrough in our understanding of the process was the function. Platelets were found to be in a preactivated state and
identification of stromal interaction molecule 1 (STIM1), were rapidly cleared from the circulation, manifesting in
described as the Ca2± sensor molecule in the ER. In 2005, macrothrombocytopenia and a bleeding phenotype. Interest-
two independent groups identified STIM1 – based on RNA ingly, Stim1Sax/+ platelets displayed a selective defect in
interference (RNAi)-based screening and knock-down – as a PLCc2-signaling, as evidenced by impaired aIIbb3 activation
conserved component of SOCE in drosophila S2 cells, HeLa and degranulation, whereas these processes were only mildly
cells and Jurkat T cell lines [38–40]. affected in response to PLCb-stimulation. These observations
STIM1 is a type I single transmembrane protein containing indicated an important function of STIM1 in platelets and
two N-terminal EF hand domains (a canonical and a hidden suggested it to be the long-sought Ca2+ sensor in these cells
one), followed by a sterile a-motif (SAM) domain, the linking store-release and Ca2+ entry. Definitive proof for this
transmembrane region, two coiled-coil region and at the C- came shortly after from studies on STIM1 knock-out mice.
terminal end a serine/proline-rich and a lysine-rich domain Parallel to the description of defective T cell and mast cell
[41,42] (Fig. 2). The EF hand domains of the molecule are function in the absence of STIM1 [44,45], STIM1-mediated
SOCE was found to be essential also for platelet function [46].
C
Intracellular Ca2+ measurements showed an almost complete
lack of SOCE in Stim1)/) platelets (Fig. 4) and severely
CC impaired Ca2+ responses to all major platelet agonists. It is
noteworthy that in accordance with the results on mast cells,
CC
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
TM obtained after passively emptying the stores with thapsigargin
(Fig. 4). Thus, STIM1 is essential for SOCE in platelets, but is
SAM also involved in the regulation of store [Ca2+] by yet-to-be
defined mechanisms.
EF hands Despite the dramatically reduced Ca2+ responses to both
N
PLCb and c activating platelet agonists, Stim1)/) platelets
showed unaltered aIIbb3 activation, aggregation and degran-
Fig. 2. Protein structure of STIM1. STIM1 is a type I single transmem- ulation through the GPCR/PLCb pathway and only mild
brane protein in the endoplasmic reticulum (ER) membrane. It bears two defects in response to GPVI/PLCc2 agonists in the absence of
N-terminal EF hand domains (a canonical and a ÔhiddenÕ one) located in
the ER lumen, which play a central role in the Ca2+ binding of the protein. flow in vitro. An explanation for this could be that 10% Ca2+
SAM, sterile a-motif; TM, transmembrane domain; CC, coiled-coil entry still occurred in these platelets (Fig. 4), which might be
domain. sufficient to support those functions. The residual Ca2+ entry

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1060 D. Varga-Szabo et al

Ca2+
α

Orai1

Orai1
Gq

Orai1

Syk

Syk
PLCγ2
PM
PLCβ
PIP2 PIP2

STIM1 Ca2+
Ca2+

IP3 R
IP 3

Ca2+ Ca2+ IP 3 IP3


R

Ca2+ Ca2+
2+
Ca2+ Ca
Ca2+ Ca2+
Ca2+ ER

Fig. 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 PLCb through Gq, whereas integrins and receptors coupled to an immunoreceptor tyrosine-based activation motif
(ITAM) activate PLCc2 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.

2000 2000
Ca2+ Ca2+
1600
TG 1600
[Ca2+]i (nM)

[Ca2+]i (nM)

TG
1200 wt 1200 –/–
TRPC1
800 800
–/–
400 Stim1 400 Orai1–/–

0 0
0 200 400 600 800 1000 s 0 200 400 600 800 1000 s

–/– –/–
Control Stim1 Orai1
400 +/+ 2000
–/–
Orai1 1600
Δ[Ca2+]i (nM)

Stim1–/–
1200
200
800

** 400
*** ***
Ca2+ – 1 mM
100 µm

Fig. 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 (D[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.

was possibly non-SOCE mediated by TRPC6 or P2X1 (see These experimental conditions mimic important aspects of
below). This rather mild activation deficit in the mutant thrombus formation in flowing blood in vivo where locally
platelets turned into a dramatically reduced capability to form produced soluble mediators are rapidly cleared. Thus, STIM1-
stable three-dimensional aggregates on collagen under high dependent SOCE appears to be of particular importance
shear conditions in a whole blood perfusion assay ex vivo. for platelet function under flow conditions; however, the

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Calcium signaling in platelets 1061

underlying mechanisms are not clear. It is also not clear why utilizing a R93W Orai1 mutant mouse line [57]. This mutation
defective SOCE has a stronger impact on platelet responses to is modeled on the mutation observed in patients with severe
GPVI agonists compared with Gq-coupled agonists as seen in combined immunodeficiency (SCID). Although similar in its
both Stim1Sax/+ and Stim1)/) mice [43,46], but it could be conclusion (e.g. Orai1 is the major SOC channel in platelets)
related to the fact that GPVI and GPCRs activate different there are some differences between the results of this study and
PLC isoforms. The low [Ca2+]i alone cannot explain the that of the Orai1)/) study. Whereas under static conditions
cellular activation defect downstream of GPVI/PLCc2 as Orai1)/) platelets show a rather mild and selective GPVI
similar or even lower levels are seen in response to thrombin signaling defect, Orai1R93W platelets show an integrin activa-
and ADP, respectively, without causing this defect. Possibly, tion defect both through GPVI and GPCRs without major
STIM1 plays a role in GPVI/PLCc2 signaling independently of differences in aggregation and thrombus formation under flow.
Ca2+ regulation, but more experiments are needed to test this One possible explanation of this is the use of slightly different
hypothesis. 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
Orai1 function.
Parallel to STIM1, calcium-release activated calcium modulator Taken together, these recent advances have now established
1 (CRACM1 or Orai1), a plasma membrane protein with four STIM1 and Orai1 as the key players of SOCE in platelets, with
predicted transmembrane domains and intracellular C- and N- STIM1 being responsible for the detection of Ca2+ release
termini, was identified as another essential component of SOCE from the intracellular stores and for regulation of Orai1, the
by the analysis of T cells from patients with severe combined major SOC channel on the platelet surface (Fig. 3).
immunodeficiency (SCID) and in Drosophila cells [47,48]. One surprising finding of the above studies is that platelets of
Shortly after, Orai1 was shown to be the pore forming unit of Stim1)/) and Orai1)/) mice, despite a virtual complete lack of
the SOC channel [49,50] and it was also shown that Orai1 agonist-induced Ca2+ entry, are still able to fulfill most of their
multimerizes with itself to create the channel [51]. In vivo studies physiological functions. Thus, Ca2+ store-release appears to be
of mast cell [52], T and B cell function [53] in Orai1 knock-out sufficient to trigger most responses such as shape change,
mice confirmed the in vitro findings. Tolhurst and coworkers integrin activation and granule release directly. Considering
showed by quantitative RT-PCR analysis of primary murine that platelets, unlike most other non-excitable cells, have to
megakaryocytes, human platelets and megakaryocytic cell lines respond very fast to activating stimuli, it is plausible that basic
that there were high expression levels of Orai1 in all three cases, cellular functions must be regulated without the delay caused
well above that of TRPC1 or other TRPCs [54]. Shortly later by an indirect process such as SOCE, which involves molecular
this was confirmed independently and the analysis of Orai1)/) coupling that requires up to 15 s to be accomplished [58]. Thus,
platelets unambiguously established Orai1 as the major SOC SOCE may be of particular relevance for processes following
channel on the platelet surface [55,56]. Using RT-PCR, human initial adhesion, primary activation and aggregation, such as
and mouse platelets were shown to express all three isoforms of thrombus stabilization, clot retraction, coagulant activity or
the Orai channel family (Orai1-3), with Orai1 being the possibly recruitment of other cells to the site of injury. In line
predominant isoform in both species, and Western-blot analysis with this notion, a major observation made in Stim1)/) and
of human platelet lysates detected strong expression of the Orai1)/) mice is reduced thrombus stability in vivo (see below)
Orai1 protein. Intracellular Ca2+ measurements on Orai1)/) and reduced PS exposure of Orai1R93W platelets [57].
platelets showed 90% reduction of SOCE (Fig. 4), which
correlates well with the results from Stim1)/) platelets [46].
TRPC1
However, in contrast to Stim1)/) platelets, Orai1)/) platelets
displayed normal Ca2+ store content and, consequently, Members of the transient receptor potential (TRP) channel
agonist-induced Ca2+ store release (Fig. 4). Therefore, family have also been suggested as candidate molecules to fulfill
Orai1)/) platelets reach consistently higher [Ca2+]i than the function of the major SOC channel in platelets. The TRP
Stim1)/) platelets in response to all tested agonists and this family can be further divided into seven subfamilies, of which
difference may be relevant for thrombus stability in vivo under the canonical TRPs (TRPCs) are the best described. Platelets
certain (experimental) conditions, such as FeCl3-induced have been shown to express members of the TRPC but also of
thrombosis [55] (Fig. 4). Furthermore, these data suggest that the TRPM subfamily [59–61]. As mentioned above, the
STIM1 contributes to the regulation of the Ca2+ store content conformational coupling model proposed TRPC1 to be a
in platelets independently of Orai1 (i.e. SOCE). Studies of major SOC channel in platelets based mainly on in vitro studies,
platelet function in Orai1)/) mice revealed a phenotype very where a blocking antibody against TRPC1 reduced SOCE in
similar to that observed in Stim1)/) mice, with only minor response to thrombin and thapsigargin [62]. The model
defects in GPVI signaling observed in the absence of flow, but suggested that Ca2+ store release triggers a de novo coupling
markedly reduced aggregate stability under flow conditions. between the IP3-R2 and TRPC1, thereby activating TRPC1 as
The function of Orai1 as the major SOC channel in platelets SOC channel in the plasma membrane to allow Ca2+ entry
has also recently been demonstrated in an independent study [36,63]. There are, however, numerous controversies in the

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1062 D. Varga-Szabo et al

literature regarding the TRPC channel family and its role in evoked by thrombin, thapsigargin or TBHQ was also reduced.
platelet function. While TRPC1 has been reported to be The reason for the discrepancies between the two studies is
expressed in lipid rafts of the platelet plasma membrane unknown and further investigation of the role of TRPC6 in
associated with TRPC4 and TRPC5 [64], others found it to be platelet physiology is required.
expressed only in internal membranes [65] and in barely
detectable amounts [65,66]. Furthermore, there are reports
P2X1
stating no effect of anti-TRPC1 treatment on thrombin- or
thapsigargin-induced SOCE [59,66]. The presence of a direct receptor-operated calcium channel on
Studies on TRPC1 knock-out mice found no significant the platelet surface was first reported in the early 1990s [69] and
differences in Ca2+ homeostasis or function of TRPC1)/) was proposed to be the P2X1 purinoceptor [70]. A few years
platelets [66] (Fig. 4), also arguing against a major role of the later, Vial et al. confirmed these results and identified the
protein for SOCE in platelets. This was further confirmed by channel unambiguously as the P2X1 receptor (Fig. 1), one of
experiments where TRPC1)/) mice were crossed with the EF seven distinct P2X receptors cloned from mammalian tissues
hand mutant Stim1Sax/+ mice, in which platelet SOC channels [71]. The natural agonist of the platelet P2X1 receptor is ATP,
are constitutively opened [43]. In this setting, lack of TRPC1 whereas ADP has been reported in a single study to have an
did not rescue the macrothrombocytopenic phenotype of the inhibitory effect [72]. Fast receptor desensitization through the
Stim1Sax/+ mice and the basal Ca2+ level in their platelets release of purines during platelet handling and the slow
remained elevated [66]. If TRPC1 was the major SOC channel recovery of the receptor made it difficult to investigate the
on the platelet surface, lack of the protein should have at least functional importance of P2X1. Despite these technical prob-
ameliorated these phenotypes. lems, it became clear from in vitro studies – using high
One explanation for the different data from previous reports concentrations of apyrase to scavenge the released ADP – that
suggesting TRPC1 to be the major SOC channel on the platelet platelet activation through the P2X1 receptor can induce
surface could be the use of antibodies of questionable selectivity reversible shape change [73] and through the PKC/Erk2
towards the protein [66,67]. A compensatory effect of other pathway contributes to complete dense granule secretion [74].
TRPCs in the TRPC1)/) mice is unlikely because TRPC6 is the This contribution occurs through the centralization of granules
only member of the TRPC channel family shown to be without release of granule content [74]. The significance of these
expressed in murine platelets and expression levels of the P2X1 mediated platelet responses seems to be the amplification
protein were unaltered. Thus, further studies will be required to of signals mediated by GPCR agonists or collagen, especially at
identify the exact role of TRPC1 in platelets. low agonist concentrations [75–77]. It is also likely that Ca2+
influx through P2X1 and the consequential membrane depo-
larization activates PLCs and IP3-receptors, thereby providing
TRPC6
further enhancement of the Gq/PLCb and GPVI/PLCc2
Besides SOCE, other Ca2+ entry mechanisms also exist in mediated activation processes [78,79].
platelets. These are the so-called non-store-operated calcium The generation of P2X1 transgenic mice provided definitive
entry (non-SOCE) mediated by DAG and a direct, receptor evidence for an important role of this ROC channel in platelet
operated calcium entry (ROCE) through the purinergic recep- function [80,81]. P2X1)/) platelets aggregated less effectively
tor, P2X1 (Fig. 1). As discussed above, PLC isoforms hydro- and showed decreased granule release in response to low doses
lyze PIP2 to IP3 and DAG. Whilst IP3 is responsible for Ca2+ of the GPVI agonist collagen, whereas they responded
release from the intracellular stores, DAG is known to directly normally to GPCR agonists and higher doses of collagen.
activate Ca2+ channels in the plasma membrane, thereby Under flow conditions, platelets from P2X1)/) blood showed
facilitating non-SOCE [4]. This mechanism exists also in reduced thrombus forming capacity at higher arterial shear
platelets and TRPC6 has been demonstrated to form a non- rates [80]. Conversely, overexpression of the receptor in mice
SOC channel [65] (Fig. 1). Unlike in the case of TRPC1, there resulted in a prothrombotic phenotype [81].
is consensus about the robust and functional expression of
TRPC6 in both human and murine megakaryocytes/platelets
Membrane Ca2+ ATPases in platelets
[60,61,64,65]. Hassock et al. proposed TRPC6 as the non-SOC
channel in platelets, based on data showing TRPC6 activation As already very small increases in [Ca2+]i lead to platelet
downstream of PLC but independent of Ca2+ release from the activation [43,73], the maintenance of a stable [Ca2+]i is
intracellular stores. They further showed that TRPC6 becomes essential to keep platelets in a resting state in the circulation,
phosphorylated by PKA and PKG; however, this phosphor- thereby preventing undesired thrombus formation. However,
ylation does not affect Ca2+ entry through the channel [65]. there is continuous leakage of Ca2+ from the intracellular
Jardin et al. in contrast suggest that TRPC6 is not only stores, which in the absence of counteracting mechanisms
involved in non-SOCE, but also in SOCE in platelets [68]. In would rapidly lead to platelet activation. Two distinct
studies where platelets were incubated with an anti-TRPC6 counteracting mechanisms have been identified in platelets:
antibody, they found that besides non-SOCE mediated by the sarcoplasmic/endoplasmic Ca2+ ATPases (SERCAs) pump
DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), SOCE Ca2+ back to the intracellular stores, whereas plasma

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Calcium signaling in platelets 1063

membrane Ca2+ ATPases (PMCAs) pump it out of the cell deficient in the protein were found to be protected in models of
[82] (Fig. 1). collagen/epinephrine-induced pulmonary thromboembolism
Platelets are known to express two SERCA isoforms and localized, laser-induced arterial thrombosis, whereas
(SERCA2b and 3) and two PMCA isoforms (PMCA1b and bleeding time was normal in most of the cases. Similarly, mice
4b) [25,27–29,83,84] but not much is known about their lacking major components of the SOCE machinery, namely
regulation and relevance for platelet function. PMCA activity STIM1 or Orai1, showed significant protection in different
in different tissues has been shown to be regulated by Ca2+/ in vivo thrombosis models due to reduced thrombus stability,
calmodulin, PKA and PKC, acidic phospholipids and prote- but only mildly prolonged bleeding times (Fig. 4). Further-
olysis (reviewed in Ref. [85]). In platelets, an inhibitory role for more, in a model system of ischemic stroke, where infarct
tyrosine phosphorylation at position 1176 of PMCA4b has development is known to be dependent on platelet function
been proposed [83], and enhanced phosphorylation of PMCA [94], both Stim1)/) and Orai1)/) bone marrow chimeras were
isoforms seems to be responsible for the observed elevation in found to be largely protected against ischemia/reperfusion
[Ca2+]i in platelets from hypertensive patients[86]. A further induced brain injury without any detectable tendency towards
study found that small GTPases of the Ras family are involved intracerebral hemorrhage [46,55] (Fig. 4). Importantly, this
in this phosphorylation process [87]. In the case of platelet protection was clinically relevant, because the global neuro-
SERCA isoforms, SERCA3b has been reported to be regulated logical and motoric functions of these animals were signifi-
by Ras-proximate (Rap) 1b [88]. In this paper the authors cantly better 24 h after ischemia than those of control mice.
report decreased SERCA3b activity as a consequence of One major limitation of the use of STIM1 or Orai1 as
decreased phosphorylation of Rap1b in spontaneously hyper- antithrombotic targets might be, however, that both are widely
tensive rats. Finally, both SERCA and PMCA activity has expressed molecules and that, as shown in the case of the Orai1
been suggested to be modulated by reactive oxygen species [89]. R93W mutation which results in SCID, mutations or lack of
There are only a few studies that address the physiological these molecules result in severe immunological deficits. There-
relevance of these two Ca2+ removal mechanisms in resting or fore cell-specific targeting of platelets is required.
activated platelets [87,90]. They came to the conclusion that in Still, these in vivo studies have revealed that the different
resting platelets the extrusion of Ca2+ through PMCAs is the Ca2+ entry mechanisms are of particular importance for
main way of regulation [87], whereas after platelet stimulation pathological thrombus formation without having a major
the activation of SERCA pumps precedes that of PMCA impact on hemostasis. This makes molecules regulating Ca2+
pumps, although the action of PMCAs lasts longer [90]. entry in platelets a promising therapeutic target for the
Because of the suicidal character of platelets, upon activation prevention and treatment of ischemic cardio- and cerebro-
they may not require fast Ca2+ export or storage conducted by vascular events.
PMCAs or SERCAs, respectively. Therefore, the above
described results on the regulation of Ca2+ ATPases in
Conclusion
platelets in vitro require in vivo confirmation. Several mouse
lines lacking individual SERCA/PMCA isoforms have been During the past 5 years, significant progress has been made in
reported in recent years (for review see Ref. [91]). However, to understanding platelet Ca2+ homeostasis in general and Ca2+
date platelet function has not been assessed in these animals. entry pathways in particular. The mechanisms regulating Ca2+
An additional way of Ca2+ removal from the platelet release from intracellular stores have been well known for some
cytoplasm has been reported in the early 1990s [92]. This is a time and now there is an increasing body of knowledge
Na+/ Ca2+ exchanger (NCX), which is able to accomplish regarding the different ways of Ca2+ influx through the plasma
very fast Ca2+ extrusion through the plasma membrane and membrane. Using the advantages of knock-out mouse models,
can also work in a reverse mode after Na+ loading of the many previous in vitro findings could be confirmed, whilst
cytoplasm. Recently, Ca2+ entry through the NCX was some others turned out to be false. The purinergic receptor
suggested to play an important role in SOCE as a consequence P2X1 has been shown to be indeed responsible for very fast
of Na+ entry through the SOC channel [93] (Fig. 1). 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
Platelet calcium entry mechanisms as potential
but also raised new ones. For example, it will be important to
therapeutic targets
clarify whether the interaction between STIM1 and Orai1 is
Ca2+ is a central and common second messenger downstream exclusive, or STIM1 also regulates other channel proteins in
of most signaling pathways in platelets. Therefore, regulators platelets. Other obvious questions remain. How is STIM1-
of Ca2+ signaling might be interesting targets for platelet Orai1 interaction regulated? Why do the two major signaling
inhibition [56]. Indeed, in vivo studies on mice lacking molecules pathways in platelets show differences in their dependency on
involved in agonist-induced Ca2+ entry have confirmed this SOCE? What role do the different PMCA and SERCA
notion in that strong antithrombotic protection but no or only isoforms play in platelets in vivo? Answering these and other
a mild hemostatic defect was seen in those animals. The platelet questions will further help to understand platelet function in its
ROC channel, P2X1, is one of the candidate molecules. Mice complexity.

Ó 2009 International Society on Thrombosis and Haemostasis


1064 D. Varga-Szabo et al

Acknowledgements 16 El-Daher SS, Patel Y, Siddiqua A, Hassock S, Edmunds S, Maddison


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for the very useful suggestions and critical discussion during membranes. Blood 2000; 95: 3412–22.
17 Newton CL, Mignery GA, Sudhof TC. Co-expression in vertebrate
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