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Review article
Transporters in human platelets: physiologic function and impact for
pharmacotherapy
Gabriele Jedlitschky,1 Andreas Greinacher,2 and Heyo K. Kroemer1
1Department
of Pharmacology, Center of Drug Absorption and Transport, and 2Department of Immunology and Transfusion Medicine,
Ernst-Moritz-Arndt-University, Greifswald, Germany
Platelets store signaling molecules (eg,
serotonin and ADP) within their granules.
Transporters mediate accumulation of
these molecules in platelet granules and,
on platelet activation, their translocation
across the plasma membrane. The balance between transporter-mediated uptake and elimination of signaling molecules and drugs in platelets determines
their intracellular concentrations and effects. Several members of the 2 major
transporter families, ATP-binding cas-
sette (ABC) transporters and solute carriers (SLCs), have been identified in platelets. An example of an ABC transporter is
MRP4 (ABCC4), which facilitates ADP accumulation in dense granules. MRP4 is a
versatile transporter, and various additional functions have been proposed, notably lipid mediator release and a role in
aspirin resistance. Several other ABC proteins have been detected in platelets with
functions in glutathione and lipid homeostasis. The serotonin transporter (SERT,
SLC6A4) in the platelet plasma membrane represents a well-characterized example of the SLC family. Moreover, recent
experiments indicate expression of
OATP2B1 (SLCO2B1), a high affinity transporter for certain statins, in platelets.
Changes in transporter localization and
expression can affect platelet function
and drug sensitivity. This review summarizes available data on the physiologic
and pharmacologic role of transporters in
platelets. (Blood. 2012;119(15):3394-3402)
Introduction
Platelets are derived from megakaryocytes. Despite being anucleate, they fulfill a multitude of functions. Platelets are not only key
players in hemostasis and in vascular disease, they also contribute
to inflammation, tumor angiogenesis, embryonic development, and
immunologic responses.1,2 Platelets contain many biologically
active molecules, such as factors triggering platelet aggregation,
growth factors, and many other compounds, which are secreted on
platelet activation. This function is highly dependent on their
intracellular compartmentalization.
Platelets contain at least 3 types of intracellular granules, in
which mediators are stored and concentrated, known as ␣,
dense, and lysosomal granules.3 The ␣-granules mainly contain
proteins critical to adhesion, such as von Willebrand factor,
thrombospondin, and fibrinogen, as well as growth factors and
protease inhibitors, clotting factors, and immunoglobulin G.
Lysosomes hold a battery of hydrolytic enzymes, which are
postulated to function in the elimination of circulating platelet
aggregates and potentially also in host defense. Dense granules,
such as lysosomes, are acidic organelles but, unlike the aforementioned organelles, contain extremely high concentrations of
small molecules, especially ADP, ATP, serotonin, and calcium.3
Dense granule molecules participate in hemostasis in numerous
ways, as ADP activates nearby platelets and serotonin causes
vasoconstriction.
The biosynthesis of platelet-dense and ␣-granules is thought to
occur in megakaryocytes through a common multivesicular intermediate body.4 However, the presence of plasma membrane
proteins, such as GPIb, in the granule membranes suggests that
these arise from both endogenous synthesis within the megakaryocyte as well as from fusion with endocytic vesicles during budding
from the plasma membrane.5
Platelet-dense granules are absent (or greatly reduced), and/or
their contents are significantly reduced in a heterogeneous group of
congenital platelets defects called ␦-storage pool deficiencies
(␦-SPD).6 These are associated with a moderate bleeding tendency.
The most severe ␦-SPD is Hermansky-Pudlak syndrome, a rare
autosomal recessive disorder in which oculocutaneous albinism,
bleeding, and lysosomal ceroid storage result from defects of
melanosomes, platelet-dense granules, and lysosomes.7,8 Mutations
in a variety of genes have been identified as causes for HermanskyPudlak syndrome, for example, in genes known to function in
vesicle trafficking and in the biogenesis of lysosome-related
organelles.9
The accumulation of compounds, such as ADP, in high concentrations inside the dense granules points to their active transport.
The identification of the ATP-binding cassette (ABC) protein
ABCC4, better known as multidrug resistance protein 4 (MRP4),
as a candidate transporter for adenine nucleotides in platelet-dense
granules,10 represents a first step toward the elucidation of this
important function of platelets. MRP4 is a markedly versatile
transporter exhibiting a broad substrate specificity composed of a
wide range of amphiphilic anions, including steroid conjugates and
eicosanoids, as well as cyclic nucleotides and nucleotide analogues.11,12 Accordingly, several other tasks have been proposed for
MRP4 in platelets taking into account that its localization can be
shifted from granules to the plasma membrane on activation of the
platelets and under certain pathophysiologic conditions.10,13,14
These include the release of lipid mediators15,16 as well as a role in
aspirin resistance under certain conditions as in patients after
coronary artery bypass graft surgery.14
Detailed experiments address the transport of serotonin in
platelets, especially its transport across the plasma membrane.
Submitted September 21, 2011; accepted February 1, 2012. Prepublished online as
Blood First Edition paper, February 14, 2012; DOI 10.1182/blood-2011-09-336933.
© 2012 by The American Society of Hematology
3394
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
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BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
TRANSPORTERS IN PLATELETS
3395
Table 1. Transporters identified in platelets
Transporter
Localization
Proposed substrates
Reference(s)
ABC transporters
ABCA7
Plasma membrane
Cholesterol, lipid mediators (?)
29,52
ABCB4 (MDR3)
Plasma membrane
Phospholipids, lipid mediators (?)
29
ABCC1 (MRP1)
Plasma membrane
Amphiphilic anions, glutathione, LTC4
29,49
ABCC3 (MRP3)
Plasma membrane
Amphiphilic anions, LTC4
29
ABCC4 (MRP4)
Dense granules, plasma membrane
Amphiphilic anions, purine nucleotide–based
10,14,15,29
mediators; glutathione, lipid mediators
SLC transporters
SLC1A1 (EAAT3)
Plasma membrane
Glutamate and neutral amino acids
19
SLC6A4 (SERT)
Plasma membrane
Serotonin, monamine transmitter
18,74,75
SLC6A12 (BGT-1)
Plasma membrane
Betaine, GABA
19
SLC18A2 (VMAT2)
Dense granules
Serotonin, monamine transmitter
85-87
SLC21/SLCO2B1 (OATP2B1)
Plasma membrane
Amphiphilic anions, statins
21
SLC23A2 (SVCT2)
Plasma membrane
Ascorbic acid
88,89
SLC35d3
Dense tubular system (?)
Nucleotide sugars (?)
90,91
SLC44A2 (CTL-2)
Granules (?)
Choline (?)
27,28
(?) indicates putative (unproven) substrate or localization.
Serotonin, which is released into circulation mainly from the
enterochromaffin cells in the gut, is rapidly taken up by platelets
and stored in platelet-dense granules, which constitute almost all
total body circulating serotonin.17 Platelets have been used as
models of neuronal transport of serotonin and also of several amino
acid transmitters for many years.18,19 Furthermore, studies of the
secretion mechanisms in platelets have indicated major similarities
to neuronal transmitter release despite the fact that these cells have
different origins.20 Neurons also contain small dense core vesicles
that contain small molecules, such as serotonin taken up and
concentrated from intracellular or extracellular pools, which resemble in many features platelet-dense granules.
An important emerging concept is that platelets may function as
“long-haul truckers” not only for endogenous biologically active
substances, such as serotonin, but also for a variety of drugs.
Although platelets are small in size (only 2.0-5.0 ␮m in diameter),3
they represent a relatively large compartment for drugs considering
their high numbers (normal range, 150-450 ⫻ 109 cells/L blood). In
adults (5 L blood), the whole body platelet volume, including
one-third splenic sequestration, accounts to approximately 20 mL
(10 fL/platelet). This potential function of platelets is not well
characterized, although it seems to be of major importance. All
systemic drugs reach their targets via the bloodstream and come
into contact with platelets; thereby, they can either be concentrated
and stored within or be excluded from platelets, depending on the
presence of uptake and export transporters in the platelet plasma
membrane. In some cases, platelets also house the target structures,
as the cyclo-oxygenase (COX)–1 enzyme,14 which is inhibited by
aspirin and other nonsteroidal anti-inflammatory drugs, or the
3-hydroxy-3-methylglutaryl coenzyme A reductase,21 which represents the target of statins. As a result of transporter-mediated
uptake, drugs can act on the target structures inside the platelets and
provoke therapeutically positive effects or negative side effects. On
the other hand, elimination of drugs from the platelet by export
pumps can limit the effect of the drug and lead to drug resistance.
Thus, because of their various functions, platelet transporters can
either serve as drug targets or as drug delivery systems, and these
functions can be greatly altered in the setting of thrombocytopenia
or with inherited or acquired platelet defects.
The purpose of this review is to examine the state of the art in
knowledge about transporters important in platelet function as well
as in pharmacotherapy.
Taxonomy of transporters
Two major protein superfamilies are involved in the transport of
drugs as well as endogenous metabolites and signaling molecules:
the ABC proteins with approximately 50 genes in humans grouped
in 7 subfamilies,22,23 and the more than 300 solute carriers (SLCs)
divided in 55 subfamilies.24,25 Members of both families have been
identified in platelets (Table 1). In general, mammalian ABC
transporters represent export pumps that bind and hydrolyse ATP,
providing the energy for a unidirectional transport of a wide range
of endogenous and exogenous compounds across membranes from
the cytoplasm to the extracellular space or into cellular organelles,
often against a concentration gradient.22,23 The typical structure of a
functional (“full-size”) mammalian ABC transporter contains at
least 2 clusters of usually 6 membrane-spanning segments as well
as 2 nucleotide-binding domains (Figure 1A).22,23 In contrast, many
SLC proteins function either by facilitating passive diffusion along
the concentration gradient of the substrate (independently of
energy input) or by cotransport and countertransport co-opting the
concentration gradient of another solute. The structures of SLCs
are often characterized by a cluster of 10 to 12 hydrophobic
membrane-spanning segments (Figure 1B).24,25
The most prominent representative of the ABC proteins is
ABCB1, better known as P-glycoprotein, which was originally
identified by virtue of its ability to confer resistance to a range of
structurally unrelated cytotoxic drugs in cancer cells (multidrug
resistance). P-glycoprotein is expressed mainly in tissues with
barrier function as in the gut or the blood-brain barrier,22 but not in
platelets. Some features of P-glycoprotein, however, are shared by
several members of the C-branch of the ABC family, the so-called
MRPs.11 Members of this subfamily, such as MRP4, which is
abundantly expressed in platelets, transport mainly amphiphilic
anions, including many drugs or drug conjugates but also a number
of endogenous signaling molecules, including arachidonate- and
purine nucleotide-derived mediators.11 Other ABC transporters,
especially of the A-branch, play an important role in the lipid
homeostasis of cells, including platelets.26
The superfamily of SLCs is composed of differed types of
carriers for a huge spectrum of substrates, including nutrients, such
as inorganic ions, sugars, amino acids, nucleotides, and vitamins,
as well as drugs. Subfamilies of SLC for which members have been
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3396
JEDLITSCHKY et al
Figure 1. Typical membrane topologies predicted for ABC and SLC transporters. (A) Structure of MRP4 (ABCC4) consisting of 2 clusters of 6 membranespanning segments and 2 regions containing nucleotide binding domains (NBDs).
(B) Structure of the serotonin uptake transport (SERT, SLC6A2) exhibiting a cluster of
12 membrane-spanning segments.
characterized within platelets include: the oligospecific Na⫹- and
Cl⫺-dependent monoamine neurotransmitter transporters (SLC6family), with the serotonin transporter (SERT, SLC6A4) as a
prominent member expressed in platelets; the vesicular amine
transporter family (SLC18); the nucleoside-sugar transporter family (SLC35); as well as the multispecific organic anion transporting
peptide family (SLC21/SLCO; Table 1). Members of the latter
family have been recognized to play an important role in the
distribution of certain drugs as statins. A member of this family,
OATP2B1 (SLCO2B1), has also been characterized in platelets.21
Another SLC protein, the choline transporter-like protein 2 (CTL-2,
SLC44A2) recently gained major attention in hematology when it
was identified to carry the human neutrophil antigen (HNA)3a, one
of the major antigens involved in transfusion-associated lung
injury.27 This protein is also expressed in platelets.28
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
2 transcript variants of the human ABCC4/MRP4 gene exist that are
predicted to encode proteins with distinct N-terminal extensions
(reference sequences NM_005845.3 variant 1 and NM_001105515.1
shorter variant 2). Analysis of the MRP4 mRNA in platelets and
mass spectrometry analysis of the protein,29 however, indicate that
the platelet 190-kDa MRP4 glycoprotein corresponds to the long
variant 1 expressed also in other tissues.30,31
The hypothesis that MRP4 is involved in the ADP storage in
platelets is further supported by studies in patients with partial
␦-SPDs, whose platelets are only deficient in ADP and not in
serotonin.13 Based on the assumption that MRP4 is essential for the
adenine nucleotide storage, one would expect that defects of MRP4
expression and localization are associated with decreased levels of
adenine nucleotides in platelet-dense granules, but normal levels of
other constituents, such as serotonin for which other transporters
are involved. Two patients were identified who exhibited this rare
phenotype of partial ␦-SPD characterized by decreased platelet
ADP content but normal serotonin levels. The MRP4 expression in
the platelets of these patients was severely diminished.13 The
underlying molecular defect leading to the diminished MRP4
expression in these platelets, however, has so far not been
identified. A similar phenotype with selective adenine nucleotide
deficiency was already previously observed in a dog model.32
Analyses of the MRP4 expression in platelets of patients with the
more prevalent classic ␦-SPD type, characterized by low adenine
nucleotide and serotonin levels, revealed a different pattern. In
these patients, MRP4 was found to be expressed in a quantitatively
normal fashion, but its localization was significantly changed
compared with normal platelets. In these patients, MRP4 seemed to
be expressed only at the plasma membrane.13
Interestingly, such a shift in the intracellular localization of
MRP4 seems to occur also under other pathologic conditions.
Recently, Mattiello et al reported that, compared with platelets
ABC transporters in platelets
Role of MRP4 (ABCC4) in platelets
The high concentration of ADP inside the platelet-dense granules
(up to 0.6M) suggests the involvement of a primary active transport
protein. A candidate protein mediating this active transport was
found in MRP4. Besides its localization in the plasma membrane,
MRP4 was demonstrated to be highly expressed in the membrane
of dense granules (Figure 2A).10,13,14 Accordingly, an altered
distribution of MRP4 was observed in platelets from a patient with
Hermansky-Pudlak syndrome in which MRP4 was only detected in
the plasma membrane because of the lack of dense granules.10 This
intracellular localization distinguishes the MRP4 expression in
platelets from that in other cell types, where it is localized mainly at
the plasma membrane. It is conceivable that different splice
variants of MRP4 are expressed in different tissues because at least
Figure 2. Proposed functions of MRP4 in mediator storage and drug resistance
depending on its localization. (A) Role of MRP4 in mediator storage and release in
normal resting and activated platelets. In resting platelets, MRP4 is mainly present in
the membrane of dense granules10,13,14 and mediates sequestration of mediators and
possibly other compounds into these organelles (left panel); in activated platelets,
MRP4 among other granule membrane proteins is inserted into the plasma membrane on granule exocytosis and may then contribute to the release of a variety of
compounds, including de novo generated lipid mediators (right panel). (B) Proposed
role of MRP4 in aspirin resistance. As suggested by Mattiello et al,14 aspirin effect on
platelets is little related to MRP4-mediated aspirin transport in normal platelets,
although MRP4 may sequestrate a part of the drug into dense granules (left panel). In
patients after coronary artery bypass graft surgery, however, MRP4 is up-regulated
on the plasma membrane already in resting platelets and mediates active extrusion of
aspirin from the cells resulting in an insufficient intracellular COX-1 inhibition by this
drug14,33 (right panel).
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BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
from healthy volunteers, platelets from patients undergoing coronary artery bypass graft surgery exhibit increased amounts of
MRP4, which was localized preferentially at the plasma membrane.14 In these patients, suboptimal platelet inhibition by aspirin
(so-called aspirin resistance) is particularly common, and Mattiello
et al hypothesized that the up-regulated MRP4 increases active
extrusion of aspirin from platelet cytosol, resulting in less effective
COX-1 inhibition14,33 (Figure 2B). This assumption is based on the
following observations: aspirin reduced uptake of Fluo-cAMP, a
potential MRP4 substrate, into dense granules of normal platelets
and is released after thrombin activation of platelets, indicating that
aspirin itself is accumulated in dense granules. Platelets from
coronary artery bypass graft patients showed a high expression of
MRP4 whose in vitro inhibition by dipyridamole or MK-571
enhanced aspirin entrapment and increased its effect on COX-1.
Platelets derived from megakaryocytes transfected with MRP4
siRNA exhibited higher aspirin entrapment.14 Further studies are
required to elucidate the role of MRP4 in aspirin transport and in
the interindividual variations of platelet inhibition by aspirin that
are also often observed in patients with acute coronary syndrome or
diabetes.33,34
When platelets are activated, granule integral membrane proteins become inserted into the plasma membrane on granule
exocytosis.3 Thus, activated platelets exhibit an additional cohort
of plasma membrane MRP4, which may alter platelet function by
increased transport of various substrates. When inserted into the
plasma membrane, MRP4 may also contribute to the export of a
variety of lipid mediators, such as thromboxane A2 and leukotrienes (Figure 2A). In contrast to ADP and serotonin, these lipid
mediators are supposed to be generated de novo on platelet
activation. Because they exert their effects mainly extracellularly
via interaction with membrane receptors, an efficient release from
the cells is also required for these mediators.
It was first recognized for the 5-lipoxygenase product leukotriene C4 (LTC4) that its biosynthesis in leukocytes is followed by a
distinct active export.35,36 MRP1 (ABCC1) was the first highaffinity transporter identified for LTC4,37and its important role in
LTC4 release from mast cells was confirmed in studies in Mrp1
knockout mice.38 ATP-dependent transport of this cysteinyl leukotriene was subsequently shown to be mediated also by other MRPs,
including MRP3 (ABCC3)39 and MRP4.15 Although platelets lack
5-lipoxygenase activity, they contain LTC4 synthase40 and can
produce LTC4 when supplied with LTA4 via transcellular mechanisms, and 3 LTC4 transporters, MRP1, MRP3, and MRP4, are
expressed in platelets, which makes it likely that platelets play an
important role in LTC4 homeostasis.
Primary prostaglandins (PGs) such as PGE2 and thromboxane
A2, have been formerly assumed to diffuse passively from the cell,
despite being poorly membrane permeable. Reid et al30 and Rius
et al41 demonstrated, however, that prostaglandins, including
thromboxane B2, are actively transported by MRP4 and that this
transport is inhibited by a number of nonsteroidal anti-inflammatory
drugs, such as ibuprofen and indomethacin.30
Another immune-modulating lipid mediator released by platelets on thrombin stimulation is sphingosine-1-phosphate (S-1-P).
Platelet-derived S-1-P modulates the chemotaxis of monocytes and
is involved in inflammatory processes.42 The release of S-1-P was
found to depend on platelet thromboxane formation and activation
of the thromboxane receptor.16 The actual release process probably
also involves a member of the MRP family.16 It was suggested that
MRP1 mediates S-1-P export in mast cells.43 Secretion of S-1-P
from human platelets was blocked by MK571,16 a cysteinyl
TRANSPORTERS IN PLATELETS
3397
leukotriene analog, which was originally identified as a specific
inhibitor for MRP137 but interferes also with MRP4.10,15 S-1-P
excretion by platelets was also inhibited by dipyridamole and
indomethacin,16 which are known to inhibit preferentially MRP4.10,30
This suggests that the release of S-1-P from platelets also involves
members of the MRP family, preferentially MRP4.
Inserted in the plasma membrane, MRP4 may also extrude
cyclic nucleotides, such as cAMP and cGMP,10-12,44 which are
both critical inhibitory intracellular second messengers regulating fundamental processes in platelets.45 The cellular levels of
these mediators are controlled by the action of phosphodiesterases, which have been also suggested as targets in antiplatelet
treatment,46 as well as by active secretion into the extracellular
space, which may provide also extracellular purine-based mediators for paracrine functions.
From a therapeutic perspective, there are thus several aspects
regarding MRP4 that provide reasons for its targeting in use of
MRP4 inhibitors for antiplatelet therapy. MRP4 transport is
inhibited by dipyridamole, which, however, has other effects, such
as inhibition of phosphodiesterases.46 More specific (but as yet
undeveloped) compounds would be a very interesting option to
interfere selectively with platelet ADP storage and possibly also
release of lipid mediators.
In addition to physiologic and pathophysiologic regulation,
genetic variations may account for variable MRP4 function. The
ABCC4/MRP4 gene is highly polymorphic with at least 25 nonsynonymous single nucleotide polymorphisms; however, the
influence of these on MRP4 expression and function in vivo
remains to be established.31 Interestingly, the ABCC4/MRP4
gene was one of the 68 genomic loci, which were identified as
putative regulators of platelet formation in a recently published
meta-analysis of genome-wide association studies for platelet
count and volume.47
Mrp4(⫺/⫺) mice have been generated and characterized mainly
with respect to their susceptibility toward nucleoside analogues.48
They exhibit no obvious bleeding tendency; however, extrapolating
these observations to humans is problematic because of interspecies differences in the properties of the transporter and in the
overall hemostatic process and compensatory mechanisms.
MRP1 (ABCC1) and MRP3 (ABCC3)
Reports on the expression of MRP1 in platelets had been controversial.15,49 The presence of MRP1 in platelet membranes could be
proven by use of 2D-nanoLC-MS/MS,29 but in a significantly lower
amount compared with MRP4. Both MRP1 and MRP4 are able to
contribute to the transport of LTC4 as well as to platelet glutathione
homeostasis because both MRPs mediate cotransport of a variety
of endogenous and exogenous compounds with reduced glutathione.11,15,50 MRP1 exports in addition oxidized glutathione (glutathione disulfate).51 Another candidate transporter for LTC4 that is
present in platelet membranes is MRP3, which also transports
cysteinyl leukotrienes, although with a lower affinity as observed
with MRP1.39 Interestingly, the expression of MRP3 and MRP4
increases during differentiation of hematopoietic progenitor cells
toward megakaryocytes, suggesting that the expression of these
proteins may be particularly important for platelet function.29
MRP1 and MRP3 are also known to confer resistance to a number
of cytotoxic agents, such as etoposide.11 Thus, they may also play a
role in resistance of platelets against toxins and can determine the
action of several amphiphilic drugs on platelet function.
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3398
JEDLITSCHKY et al
ABCA and ABCB transporters
Transcripts of several members of the ABCA family, such as
ABCA3, ABCA4, ABCA6, ABCA7, and ABCA9, have been
detected in human platelets.29 ABCA7 was shown to be preferentially expressed in platelets and localized at the plasma membrane
of rat and human platelets.29,52 ABCA proteins are typically
associated with lipid translocation processes in membranes. Two
types of intramembrane lipid translocators can be defined: flippases,
which translocate lipids from the outer leaflet to the inner leaflet of
the membrane; and floppases, which mediate the reverse process
(ie, translocation from the inner to the outer leaflet of the membrane
bilayer).53 Such membrane remodeling processes also play an
important role in platelet function, especially during secondary
hemostasis. The flopping of phosphatidylserine from the inner to
outer membrane leaflet during platelet activation, together with
microparticle shedding, provides a catalytic surface for the assembly of coagulation complexes.54,55 There is a rare platelet defect, the
Scott syndrome,56 which underscores the importance of this
pathway. In these patients, although primary hemostatic function of
platelets is intact, the platelets do not support the assembly of
coagulation complexes because of defective scrambling of membrane phospholipids.56 In addition, there exists an inverse condition, the Stormorken syndrome, in which platelets constitutively
expose phosphatidylserine on the external leaflet of their plasma
membrane.57
Two protein classes are thought to be involved in membrane
remodeling: a nonspecific and energy-independent family of socalled scramblases58 and ABC transporters, especially of the A- and
B-branch. ABCA1, which is involved in cholesterol efflux from
cells and in phagocytosis, and which is mutated in Tangier
dyslipidemia (absence of high-density lipoprotein-cholesterol from
plasma),59,60 has been considered also to play a role in Scott
syndrome. This hypothesis is based on the observation that targeted
deletion of the corresponding gene locus resulted in a phenotype
evocative of partial Scott syndrome,61 and a mis-sense mutation in
the ABCA1 gene was identified in a Scott syndrome patient.62
However, the expression of ABCA1 in platelets has not been
established so far.
ABCA7, which has been identified in platelets, however, shares
several features with ABCA1. ABCA7 also mediates the formation
of high-density lipoprotein when exogenously transfected and
expressed and was also reported to relate to the phagocytotic
function of cells.63 Genetic variants of the ABCA7 gene have been
found to be associated with Alzheimer disease.64 ABCB4, also
known as MDR2/3 (Mdr2 in rodents, MDR3 in humans), represents another floppase identified in the platelet plasma membrane.29
This finding is surprising because the expression and function of
MDR3 were thought to be limited to the canalicular membrane of
hepatocytes.65 Here, MDR3 “flops” phosphatidylcholine from the
inner to the outer leaflet of the canalicular membrane, thereby
making this phospholipid available for extraction into the canalicular lumen by bile salts.66 Mutations in the ABCB4/MDR3 gene
cause progressive familial intrahepatic cholestasis.66 However, so
far none of the ABCA and ABCB transporters has been explicitly
confirmed as a floppase for PS. The interaction of platelet
microparticles with the endothelium and leukocytes in addition
triggers inflammation. Thus, the lipid translocators in platelets may
have an important role in thrombosis and in inflammation.
Besides their role in plasma membrane remodeling, MDR3 and
ABCA proteins may be involved in the release of lipid mediators as
lysophosphatidic acid and S1-P from platelets. Lysophosphatidic
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
acid is a bioactive lipid that binds to cell surface G-protein-coupled
receptors to regulate cell growth, differentiation, and development.67 In the cardiovascular system, lysophosphatidic acid alters
the endothelial barrier function and is a weak platelet agonist.68,69
In addition, it was proposed that members of the ABCA subfamily
are involved in the transport of S-1-P,52 especially ABCA7.
Furthermore, ABCAs and MDR3 may function in the defense of
platelets against highly lipophilic compounds.
SLC transporters in platelets
Serotonin transporters
Serotonin (5-hydroxytryptamine [5-HT]) is best known for its role
as neurotransmitter involved in mood disorders.18 However, serotonin is also stored in platelets and, when released, promotes
platelet aggregation via the serotonin receptor (5-HT2A) on platelets.17 Moreover, it exhibits strong vasoactive properties, possibly
through stimulation of serotonin receptors on endothelial cells and
through nitric oxide production.70 Recently, it was also shown that
serotonin strongly induces extracellular matrix synthesis in interstitial fibroblasts and promotes tissue fibrosis.71
Serotonin storage in platelets results from a 2-step process:
(1) the uptake across the platelet plasma membrane; and (2) the
transport across the dense granule membrane. The uptake of
serotonin into the platelet cytosol is mediated by SERT (also called
5-HTT). The cDNA of human SERT has been cloned, and the gene
(SLC6A4) has been assigned to the human chromosome 17.72,73
Expression of SERT was characterized in different tissues, including brain and platelets. Thereby, the transport protein appears
identical in brain and platelets.74 SERT belongs to the SLC6 gene
family of Na⫹/Cl⫺-dependent neurotransmitter transporter proteins.75 The actual uptake process involves the binding of serotonin
to its recognition site within the transporter and its transport across
the membrane together with an Na⫹ ion. A second step involves the
translocation of a K⫹ ion across the membrane to the outside of the
cell. This requirement for K⫹ countertransport is unique for SERT
within the SLC6 family.76 Because both tricyclic antidepressants
and the newer selective serotonin reuptake inhibitors (SSRIs) bind
to SERT and inhibit serotonin uptake, the importance of understanding the biochemical characteristics of this transporter has long been
appreciated.18 SSRIs block the reuptake of serotonin into neurons
as well as the uptake into platelets; thus, platelets were used as
model for monitoring the effect of antidepressants.77,78
In addition, platelets exhibit uptake mechanisms for several
amino acid transmitters, among them ␥-aminobutyric acid, glutamate, aspartate, and glycine, and characteristics of the platelet
uptake functions resemble those of the uptake in the central
nervous system,19 qualifying platelets as a favored model system in
neurology (Figure 3).
Prolonged intake of SSRIs can lead to a significant decrease in
platelet serotonin.17,77 However, bleeding problems are rarely
observed in SSRI-treated patients. This may be the result of the
postulated prothrombotic disturbance in untreated depression accompanied with an increased cardiovascular risk.79 Although under
normal conditions the bleeding risk induced by SSRIs is low, they
can increase the risk for bleeding in major surgery.80 Serotonin
promoter polymorphisms have been linked to major depressive
disorders as well as to an increased risk of new cardiac events after
acute myocardial infarction.81-83 SERT knockout mice have been
generated and are vital, but platelet function has not been studied in
detail.84
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BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
TRANSPORTERS IN PLATELETS
3399
OATPs
Figure 3. Transport and storage of monoamine mediators in neurons and
platelets. SERT mediates reuptake of serotonin (5-HT) in neurons as well as uptake
into platelets and is inhibited by tricyclic antidepressants and SSRIs.18,79 The storage
in dense vesicles is supposed to be mediated by the VMAT (SLC18A).85-87 The
energy for this transport is provided by the function of the vacuolar H⫹-ATPase.
5-HT-R indicates serotonin receptor.
Less is known about the transport of serotonin across the dense
granule membrane, which is supposed to be mediated by a
reserpine-sensitive vesicular monoamine transporter (VMAT,
SLC18), which is also present in the secretory vesicles of monoaminergic neurons and neuroendocrine cells in the gut.85,86 This
transport is driven by an electrochemical proton gradient across the
vesicular membrane, which is generated by the vacuolar H⫹ATPase and is potently inhibited by reserpine and tetrabenazine.85,86 In mammals, 2 closely related isoforms of the monoamine
transporter, termed VMAT1 and VMAT2 (SLC18A1 and
SLC18A2), respectively, have been identified, which are located on
different vesicle subtypes.85,86 VMAT2 has been shown to be
expressed mainly in synaptic dense core vesicles, which resemble
platelet-dense granules.87 Both VMATs transport serotonin, dopamine, epinephrine, and norepinephrine but differ in their substrate
preferences and affinities.
Transporters for nutrients and metabolic intermediates
Platelets, like other cells, need uptake transporters for nutrients.
For example, platelets accumulate ascorbic acid through the
expression of SVCT 2 (SLC23A2), a member of the Na⫹dependent ascorbic acid transporter family (SLC23).88 Interestingly, platelets can compensate for fluctuations in ascorbate
levels by modulating the expression of SVCT2 at the translational level.89 Platelets, although anucleate, contain RNA, some
of which is translated into proteins, including transporters,
according to requirements.89 SLC proteins also often transport
metabolites from the cytosol into intracellular organelles. Interestingly, mutations in the Slc35d3 gene have been associated
with platelet-dense granule defects in a murine model.90 Slc35d3
is a member of the SLC35 nucleotide sugar transporter family.
Members of this family are characterized as antiporters, transporting nucleotide sugars from the cytosol into the lumen of the
Golgi apparatus and/or the endoplasmic reticulum.91 In platelets, the dense tubular system represents residual endoplasmic
reticulum of the parent megakaryocytes. However, the actual
localization of this transporter in platelets as well as its role in
platelet-dense granule dysfunction has not been further
elucidated.
Nutrients and drugs can share common transporters. The relevance
of platelet uptake transporters for the distribution and action of
drugs has been well demonstrated for statins. Available evidence
supports the notion that the beneficial effects of statins on
hypercholesterolemia-associated diseases, such as acute coronary
syndrome, stroke, and atherosclerotic lesions, are attributable not
only to their low-density lipoprotein-lowering effect but also to
additional mechanisms of action.92 These “pleiotropic” effects
include the stabilization of arterial plaques, normalization of
endothelial functions, anti-inflammatory effects, and inhibition of
platelet thrombus formation.93 On the molecular level, pleiotropic
effects of statins have been attributed to the inhibition of 3-hydroxy3-methylglutaryl coenzyme A reductase in nonhepatic structures.94
This inhibition results in diminished biosynthesis of mevalonate,
the precursor to cholesterol, and also to hydrophobic prenyl
moieties, which are essential for proper sorting and function of
several cell membrane-associated proteins, as receptors.95 If the
modification of platelet function by statins proceeds via inhibition
of platelet 3-hydroxy-3-methylglutaryl coenzyme A reductase,
uptake of these drugs into platelets is a prerequisite. In the liver, the
prime target organ for statins, uptake of statins, was shown to be
mediated by transporters of the organic anion-transporting polypeptide (OATP/SLCO) family, mainly OATP1B1.96 A related transporter, OATP2B1, was detected in platelets and localized to the
plasma membrane.21 OATP2B1 was shown before to transport
atorvastatin and rosuvastatin as high-affinity substrates.97,98 Accordingly, an active transport of atorvastatin into platelets could be
demonstrated, which was inhibited by the known OATB2B1
substrate estrone sulfate and vice versa.21 As a consequence of
OATP2B1-mediated uptake of atorvastatin, a significant reduction
of thrombin-induced Ca2⫹ mobilization in platelets was observed,
which could be mechanistically explained by reduced prenylation
of signal proteins. The effect was reversed by addition of mevalonate, the precursor to prenyl moieties as well as in presence of
estrone sulfate, which competitively inhibits atorvastatin uptake.21
Besides statins, drugs and metabolites such as dehydroepiandrosterone-3-sulfate or estrone sulfate, which are described as
OATP2B1 substrates, could be taken up into platelets by this
transporter. For example, dehydroepiandrosterone-3-sulfate administration improves platelet superoxide dismutase activity, which
protects cells against oxidative damage.99 The expression of
OATP2B1 is probably only one example of how transporters in
platelets influence effects of drugs, or vice versa, how drugs affect
platelet biology. The list of drug uptake transporters in platelets is
expected to grow rapidly, as this aspect of platelet transporters is
addressed more intensively.
In conclusion, platelets contain a variety of transporters in their
plasma membrane as well as within the membranes of their
intracellular compartments (summarized in Figure 4). These proteins play a vital role in the storage and release of endogenous
signaling molecules, implicating them as relevant structures for
certain platelet function disorders as it has been shown for storage
pool deficiencies. Platelets may serve also as vehicles that pick up
compounds from various regions in the organism, including the
gut, and transport them to their target organs. This would add new
aspects to the role of platelets in many disorders, including host
defense and immunology.100
Transporters are also potential new targets for pharmacologic
inhibition of platelet function. Inhibiting a transporter instead of a
receptor provides attractive options. On the one hand, the effects
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JEDLITSCHKY et al
capacity. A better understanding of these mechanisms may help to
recognize potential prothrombotic or prohemorrhagic effects of
drugs. Finally, platelets may influence pharmacotherapy of possibly many drugs and accordingly contribute to interindividual
variation in drug response. Therefore, a detailed understanding of
the expression, function, and regulation of transporters in platelets
will add to a better understanding of platelet biology and in many
ways may result in improved therapeutic options.
Acknowledgments
Figure 4. Platelet transporters and their proposed functions at a glance.
Platelets express a variety of SLC and ABC transporters, which are located in the
plasma membrane as well as in the membranes of intracellular compartments,such
as the dense (␦) and ␣ (␣) granules, and in other intracellular membrane systems,
such as the dense tubular system (DTS; for details and references, see Table 1).
They play a vital role in the uptake, sequestration, and release of mediators involved
in platelet function. With respect to pharmacotherapy, the platelet represents a
pharmacokinetic microcompartment, in which the interplay between uptake and
elimination transporters determines intracellular drug concentrations.
The authors thank Theodore Warkentin (Department of Pathology
and Molecular Medicine, McMaster University, Hamilton, ON) for
expert proofreading of the manuscript.
This work was supported by Deutsche Forschungsgemeinschaft, Germany (grants JE234/4-1 and SFB/TR19), and by the
GANI-MED project (the Greifswald Approach to Individualized
Medicine), funded by the Federal Ministry of Education and
Research, Germany; and by the Center for Immune Reactions in
Cardiovascular Disease, funded by the Federal Ministry of Education and Research, Germany.
Authorship
are long lasting and relatively robust even in patients with moderate
compliance; and on the other hand, the drug effect can be
counteracted immediately by transfusion of platelet concentrates,
as the transfused platelets contain the substrates of the respective
transporter. From a more pharmacologic point of view, platelets
represent pharmacokinetic micro-compartments, in which the
combination of uptake and elimination transport, possibly together
with intracellular metabolism, determines pharmacokinetics of
drugs. Transporters can be specifically used to direct antiplatelet
drugs to target structures inside the platelets. Probably more
important, many non-antiplatelet drugs may interfere with the
physiologic substrates of these transporters in platelets and may in
this way affect platelet function and modulate their hemostatic
Contribution: G.J. wrote the paper and designed the figures; and
A.G. and H.K.K. edited the manuscript and provided conceptual
insights.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Gabriele Jedlitschky, Institut für Pharmakologie, Center of Drug Absorption and Transport, Ernst-Moritz-ArndtUniversität Greifswald, D-17487 Greifswald, Germany; e-mail:
[email protected]; and Andreas Greinacher, Institut für
Immunologie und Transfusionsmedizin, Ernst Moritz-ArndtUniversität Greifswald, 17487 Greifswald, Germany; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2012 119: 3394-3402
doi:10.1182/blood-2011-09-336933 originally published
online February 14, 2012
Transporters in human platelets: physiologic function and impact for
pharmacotherapy
Gabriele Jedlitschky, Andreas Greinacher and Heyo K. Kroemer
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