J. of Cardiovasc. Trans. Res.

(2013) 6:299309
DOI 10.1007/s12265-012-9445-9

Platelet Biology and Receptor Pathways

Giovanni Cimmino & Paolo Golino

Received: 19 November 2012 / Accepted: 27 December 2012 / Published online: 10 January 2013
# Springer Science+Business Media New York 2013

Abstract The main function of platelets is to participate in Keywords Platelet . Aggregation . Membrane receptors .
primary hemostasis through four distinct steps: adhesion, Coagulation
activation, secretion, and aggregation. Unraveling the mo-
lecular mechanisms underlying these steps has led to a better
understanding of the pathophysiology of bleeding disorders, Introduction
on one hand, and of thrombotic diseases, such as acute
coronary syndromes, on the other. Platelets are cytoplasmic Under normal conditions, platelets circulate freely in the
fragments of megakaryocytes formed in the bone marrow. blood without interacting with each other or with the vas-
They lack nuclei but contain organelles and structures, such cular endothelium. In the presence of endothelial damage,
as mitochondria, microtubules, and granules. Platelet gran- whether from a vascular injury or rupture of an atheroscle-
ules contain different bioactive chemical mediators, many of rotic plaque, a chain of events is triggered, ultimately lead-
which have a fundamental role in hemostasis and/or tissue ing to platelet-rich clot formation. Depending on the
healing. The platelet cytoplasm contains an open canalicular initiating event, this may represent normal hemostasis or
system that increases the effective surface area for the intake pathologic intravascular thrombosis [1].
of stimulatory agonists and the release of effector substan-
ces. The submembrane region contains microfilaments of
actin and myosin that mediate morphologic alterations char-
Platelet Morphology
acteristic of shape change. Resting platelets remain in the
circulation for an average of approximately 10 days before
Platelets are anucleated cytoplasmic fragments originating
being removed by macrophages of the reticuloendothelial
from megakaryocytes in the bone marrow; they form as a
system. A wide variety of transmembrane receptors cover
result of cytoplasmic divisions not associated with concom-
the platelet membrane, including many integrins, leucine-
itant nuclear division (a process called endomitosis) of
rich repeat receptors, G protein-coupled receptors, proteins
megakaryocytes in response to thrombopoietin [2]. After
belonging to the immunoglobulin superfamily, C-type lectin
leaving the bone marrow, platelets circulate in the blood
receptors, tyrosine kinase receptors, and a variety of other
for about 10 days. Their volume is about 7 m3 with a
types. In this article, we will review platelet biology under
discoid shape and a major diameter of about 300 nm. A
physiological and pathological conditions, with particular
complex internal system of microtubules and actin cytoskel-
emphasis on the function of their membrane receptors.
eton is responsible for shape change modifications after
activation.
G. Cimmino : P. Golino
The resting platelet can be divided into three zones: (a)
Section of Cardiology, Department of Cardio-Thoracic and
Respiratory Sciences, Second University of Naples, Naples, Italy peripheral zone: responsible for adhesion and aggregation,
consists of a fluffy glycocalyx coat, platelet membrane, and
P. Golino (*) cytoskeleton; it may contain absorbed coagulation factors I,
Division of Cardiology, Second University of Naples, c/o
V, VIII, XI, and XII, receptors for adenosine diphosphate
Ospedale S. Sebastiano Via Tescione, 1,
81100 Caserta, Italy (ADP), thrombin, von Willebrand factor (vWF), collagen,
e-mail: paolo.golino@unina2.it fibrinogen, fibrin, fibronectin, epinephrine, platelet-
300
activating factor, thrombospondin (TSP), thromboxane A2
(TXA2), prostacyclin, epinephrine, serotonin, and glycosyl-
transferase [3]; (b) solgel zone: responsible for contraction
and support of the microtubule system; it contains the con-
necting system called the open canalicular system and the
dense tubular system; and (c) organelle zone: contains the
dense body system, nonmetabolic ADP, serotonin, catechol-
amines, calcium, -granules, platelet factor 4 (PF4), platelet
mitogenic factors (such as platelet-derived growth factor
[PDGF]), fibrinogen, -thromboglobulin, lysosomal gran-
ules, mitochondria, and glycogen granules [46].
Three specific granule populations (dense granules, lyso-
somes, and -granules) store different types of constituents,
some of which are at high concentrations. Each granule
population has specific properties concerning both the struc-
ture and the role played by the released constituents. Dense
granules contain small nonprotein molecules that are secret-
ed upon platelet activation. The -granules are the most
abundant, comprising roughly 10 % of the platelet volume,
10-fold more than dense granules. They contain large adhe-
sive and healing proteins, such as hemostatic factors (e.g.,
factor V, vWF, fibrinogen), angiogenic factors (e.g., angio-
genin, vascular endothelial growth factor [VEGF]), antian-
giogenic factors (e.g., angiostatin, PF4), growth factors
(e.g., PDGF, bFGF, SDF1), proteases (e.g., MMP2,
MMP9), necrotic factors (e.g., TNF, TNF), and other
cytokines. Among the platelet-specific proteins are several
peptides that modulate cell growth. Of these, PDGF is one
of the most extensively studied [7]. Lysosomes contain
hydrolases, the function of which is to eliminate circulating
platelet aggregates.
Platelet Function
The main physiological role of platelets is to contribute to
primary hemostasis, a defense mechanism aimed at prevent-
ing blood loss when the continuity of the vasculature is
interrupted. In addition to their critical role in hemostasis,
new evidence suggests that platelets may exert other func-
tions in regulating the immune response, promoting inflam-
mation, and cancer metastasis [9].
Classically, the complex series of biochemical and cellu-
lar processes responsible for the biological functions of
platelets can be divided into four different steps: adhesion,
activation, secretion, and aggregation (Fig. 1).
Adhesion
Adhesion and aggregation should be considered as distinct
processes through which platelets establish individual con-
tacts with extracellular surfaces or stick to one another
forming aggregates, respectively.
J. of Cardiovasc. Trans. Res. (2013) 6:299309
The initial tethering of platelets at sites of vascular injury
is mediated by glycoprotein (GP) Ib/V/IX, a structurally
unique receptor complex expressed in megakaryocytes and
platelets. vWF is the major ligand for one component of this
complex, GPIb [1, 10, 11], and its absence causes important
defects in primary hemostasis and coagulation. Besides
GPIb, several collagen receptors with a tethering function
are found on the platelet surface, notably GPVI and GPIa/IIa
(or 21 integrin), members of the immunoglobulin super-
family [1, 10, 11]. GPIV, a non-integrin protein, may also
play a role in plateletcollagen interactions, as well as acting
as a receptor for TSP [10, 11]. Other integrins that contribute
to platelet adhesion include GPIc/IIa (a51), a fibronectin
receptor, a laminin receptor, and a vitronectin receptor that
also recognizes many of the ligands that bind to the GPIIb/
IIIa receptor [11].
Shear stress is an important factor that modulates platelet
function. Shear stress is related to flow rate, diameter of the
vessel, and fluid viscosity [12]. Blood circulating in vessels is
exposed to shear stress caused by the force necessary to
produce flow; the difference in velocity between layers situ-
ated at varying distances from the vessel wall determines the
shear rate, which is directly proportional to the shear force and
inversely proportional to the viscosity of blood. Studies per-
formed under controlled conditions of high shear stress, to
mimic the rheological situation existing in certain districts of
the arterial circulation, have suggested that shear stress may
influence platelet aggregation through different adhesive
ligands, including vWF, GPIb/IX, and GPIIb/IIIa [13, 14].
Platelets can directly or indirectly interact with collagen
through their surface receptors: GPIb/V/IX complex, GPVI,
and GPIa/IIa (or integrin a2b1). Under high shear flow
conditions (i.e., arterial blood flow), the initial interaction
is indirect and mediated by the interposition of vWF, a
circulating protein which is able to bind exposed collagen.
As a consequence of this binding, vWF switches from its
inactive to its activated conformation [15]. The GPIb/V/IX
complex interacts with activated vWF immobilized on ex-
posed collagen, and the fast on and off rates of this interac-
tion causes rolling of platelets [16]. This process does not
mediate firm adhesion of the cells, but reducing platelets
velocity, allows platelets to bind directly to collagen through
its two chief collagen receptors, GPVI and GPIa/IIa, stabi-
lizing the growing thrombus [17]. GPIa/IIa (or integrin
21) is the second most important platelet integrin (next
to IIb3). It is expressed on resting platelets surface in
low-affinity state for collagen, but platelets activation shifts
the receptor to a high-affinity state [18].
Activation
Platelet activation follows adhesion and can be initiated by
several mechanical and chemical stimuli. Adhesion of
J. of Cardiovasc. Trans. Res. (2013) 6:299309
Fig. 1 Schematic view of
platelets function (see text for
details)
platelets to collagen and other components of the subendo-
thelial matrix (collagen, vWF) are among the strongest
stimulators of platelet activation. The activation of platelets
is associated with stimulation of several metabolic path-
ways, changes in the shape of platelets, and secretion [1].
Binding of collagen to GPVI, a type I transmembrane
protein belonging to the Ig superfamily, plays a central role
in platelet activation through multiple intracytoplasmic path-
ways (i.e., elevation of intracellular Ca2+, phosphoinositide
metabolism, phosphorylation of cytoplasmic and nuclear pro-
teins); this results in the conversion of platelet shape from
discoid to spherical and degranulation [19]. During platelet
shape change, the discoid cells undergo cytoskeletal modifi-
cations, including the disassembly of a microtubule ring that
results in an intermediate spherical shape. This is followed by
actin polymerization and the slower extension of filopodia [1].
Secretion
Following activation, platelets release the substances stored
in their specific granules. This release reaction is an
important step of primary hemostasis. Energy required for
platelet reactivity is provided by the mitochondria [3]. Once
activated, platelets readily release the constituents present in
their cytoplasmic granule, such as ADP, ATP, serotonin,
TXA2 [20].
Aggregation
Irrespective of the agonist, the final step after activation is
the formation of platelet aggregates. This event is mediated
by the binding of adhesive proteins to the now ligand-
receptive form of the GPIIb/IIIa receptor. It is well-known
that activation causes changes in the shape of platelets and
conformational changes in GPIIb/IIIa receptors, transform-
ing the receptors from a ligand-unreceptive to a ligand-
receptive state [21]. The receptive form of GPIIb/IIIa binds
301
fibrinogen molecules, which form bridges between adjacent
platelets and facilitate platelet aggregation [22]. Inhibitors of
GPIIb/IIIa receptors also bind to these receptors, blocking
the binding of fibrinogen and thus preventing platelet ag-
gregation [2124]. Other adhesive GPs, including fibronec-
tin, vWF, and vitronectin, also bind to these receptors. As a
result of multiple reactions of this type, platelets become
aggregated into a hemostatic plug.
Platelet Membrane Receptors
Two ADP receptors are expressed in platelets: P2Y1, which
is coupled to Gq and contributes to initial aggregation, and
P2Y12, which is coupled to G12 and decreases intracellu-
lar levels of cyclic adenosine monophosphate (cAMP). The
P2Y1 is a purinoceptor widely distributed in many tissues
including the heart, blood vessels and blood cells, neural
tissue, testis, prostate, and ovary [25, 26]. About 150 P2Y1
receptor-binding sites are expressed per platelet [27]. The
P2Y1 receptor triggers the mobilization of calcium from
internal stores, which results in platelet shape change and
a weak, transient aggregation in response to ADP. As a
consequence, at the intracellular level, the calcium signal
is abolished, while the ability of ADP to inhibit cAMP
formation is preserved [28, 29]. The P2Y1 receptor also
participates in the aggregation response to collagen and
plays a key role in collagen-induced shape change when
TXA2 formation is prevented [30].
The P2Y12 receptor is coupled to the inhibition of
adenylyl cyclase activity through the activation of a Gi2
G protein subtype, which is a critical component of the
signaling pathway for integrin IIb3 activation [31].
However, adenylyl cyclase inhibition resulting in lowering
cAMP levels is not sufficient to cause platelet aggregation
per se [32]; thus, other signaling events are required for full
activation of the IIb3 integrin and subsequent aggregation.
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One important intracellular pathway, which regulates Gi-
dependent integrin IIb3 activation, is represented by phos-
phoinositide 3-kinase (PI3K). The PI3K isoform p110 reg-
ulates integrin activation through a classical lipid kinase-
dependent mechanism, involving the small GTPase Rap1
and/or the serinethreonine protein kinase B/Akt (PKB/Akt)
[33], whereas p110 appears to regulate integrins principally
through a noncatalytic signaling mechanism [34].
Coactivation of the P2Y1 and P2Y12 receptors is necessary
for normal ADP-induced platelet aggregation, since separate
inhibition of either of them with selective antagonists results
in a dramatic decrease in aggregation [35].
P2Y12 receptor signaling also stimulates surface expres-
sion of P-selectin and secretion of TXA2 [36]. TXA2 is
produced de novo and, like ADP, is released by activated
platelets. It is enzymatically generated from arachidonic
acid (AA) by cyclooxygenase-1 (COX-1) and TX synthase.
TXA2 binds platelet receptors TP and TP which differ in
their cytoplasmic tail; however, its effects in platelets are
mediated primarily through TP [37]. TP and TP couple
to the proteins Gq and G12 or G13, all of which activate
phospholipase C. This enzyme degrades the membrane
phosphoinositides (such as phosphatidylinositol 4,5-
bisphosphate [PIP2]), releasing the second messenger ino-
sitol triphosphate (IP3) and diacylglycerol (DAG). DAG
activates intracellular protein kinase C, which causes protein
phosphorylation. The release of IP3 increases cytosolic lev-
els of Ca2+, which is released from the endoplasmic reticu-
lum. Both ADP and TXA2 are secreted from adherent
platelets, contribute to the recruitment of circulating plate-
lets, and promote alterations in platelet shape and granule
secretion.
Thrombin is the ultimate effector of the coagulation
cascade and, in addition to cleaving fibrinogen and other
circulating substrates, it acts as a circulating mediator able to
induce different cellular responses in platelets, endothelial
cells, and SMCs. Thrombin binds a G protein-coupled class
of receptor named protease-activated receptors (PARs) [38,
39]. Four subtypes of PARs have been identified in humans,
which are widely distributed among different cell types and
tissues. PAR-1, PAR-3, and PAR-4 are activated by throm-
bin, whereas PAR-2 can be activated by trypsin, tryptase,
and coagulation factors VIIa and Xa [8, 9], but not by
thrombin. PAR-1 can be considered as the prototype of this
receptor family. It is converted in the active form by the
cleavage of its N-terminal exodomains at a specific site
(thrombin cleavage site) that unmasks a new N terminus
domain (SFLLRN) able to bind intramolecularly to the body
of the receptor, resulting in a transmembrane signaling via G
protein activation [40]. Thus, PAR-1 carries its own silent
agonist peptide and achieves a sort of self-activation pro-
cess after thrombin cleavage. Each of the four PARs is
activated via this mechanism, with some minor variations
J. of Cardiovasc. Trans. Res. (2013) 6:299309
in each case. The cleavage process is irreversible, thus PAR-
1 pathway needs several inactivating mechanisms to switch
off signal transduction: receptor desensitization (via phos-
phorylation for PAR-1, but not for PAR-4) [8, 44], receptor
internalization, and lysosomes degradation [41] are the main
known mechanisms.
Thrombin-mediated platelet activation in humans occurs
through the activation of PAR-1 and PAR-4. PAR-1 is con-
sidered the principal proteaseplatelet receptor and mediates
the activation of human platelets at low (<1 nM) thrombin
concentrations; PAR-4 necessitates higher (>30 nM) concen-
tration of thrombin [42]. PAR-1 signaling in platelets results in
heterotrimeric G proteins activation (G12/13, Gq, and
Gi/z) with consequent TXA2 production, ADP release, mo-
bilization of P-selectin and CD40L, integrin activation, and
platelet aggregation. PAR-1, in addition, enhances thrombin
formation and fibrin generation inducing platelets procoagu-
lant activity [43]. Recently, several studies also showed a
cross-reaction of P2Y12 and PAR-1 pathways in mediating
platelets activation. Despite the lower affinity of PAR-4 for
thrombin, both PAR-1 and PAR-4 contributes to platelet acti-
vation. PAR-4 is activated, shut off, and internalized more
slowly than PAR-1 and produces the majority of the integrated
thrombin-triggered calcium signals of the two receptors. In
addition, PAR-1 and PAR-4 counter-regulate the release of
endostatin and VEGF from human platelets [44]. The impor-
tance of these differences in PARs signaling in human platelet
is still not clear. The current hypothesis is that PAR-4 acts as
an auxiliary receptor mediating thrombin signaling to distinct
effectors (i.e., release of distinct subsets of platelets granules)
or with different kinetics compared with PAR-1. A schematic
view of the major platelet receptors with its own function is
reported in Table 1.
Regulation of Platelet Function: The Novel Role
of microRNAs (miRNA)
Platelets activation is a highly regulated process. Despite the
fact that they are anucleate cell fragments, it has been shown
that platelets contain a number of megakaryocyte-derived
mRNAs [45] encoding for different proteins involved in
metabolism, signaling, inflammation, and immunity [46].
Platelets also contain rough endoplasmic reticulum and pol-
yribosomes, enabling them to synthesize different proteins
[47]. These mechanisms allow platelets to modify their
protein expression and, as a consequence, their functions
[48]. For example, specific mRNA translation may vary in
clinical conditions, such as acute myocardial infarction, as
recently reported [49]. Identification of disease-associated
platelet-specific transcripts is of particular relevance in
platelet pathophysiology, since it may lead to the discovery
of novel therapeutic targets.
J. of Cardiovasc. Trans. Res. (2013) 6:299309 303

Table 1 Major Platelet Mem-

brane Receptors Name Type Agonists Role

glycoprotein Ib (GPIb) Integrin vWF Adhesion

glycoprotein VI (GPVI) non-integrin collagen Adhesion
glycoprotein Ia/IIa (GPIa/IIa) integrin collagen Adhesion
glicoprotein Ib-V-IX complex (GPIb-V-IX) integrin collagen Adhesion
glycoprotein VI (GPVI) non-integrin collagen Activation
P2Y1 G protein-coupled receptor ADP Aggregation
P2Y12 G protein-coupled receptor ADP Aggregation
Thromboxane Receptor (TP) G protein-coupled receptor TXA2 Aggregation
Protease Activated Receptor (PAR) G protein-coupled receptor Thrombin Aggregation
glycoprotein IIb/IIIa (GPIIb/IIIa) integrin Fibrinogen Aggregation

The evidence that platelets contain mRNAs and are ca-
pable of protein synthesis has raised the issue of how these
mRNAs are regulated. Among the various mechanisms in-
volved, mRNA splicing [50, 51] and miRNAs are of great
relevance. miRNAs are a class of small noncoding RNAs
(approximately 21 to 23 nucleotides) which function pre-
dominantly as sequence-targeted modifiers of gene expres-
sion acting on posttranscriptional processes, such as mRNA
degradation and translational repression. They originate
from longer precursor RNAs (100 to 1,000 nucleotides
primary miRNAs) that are processed in the nucleus into a
70- to 100-nucleotide hairpin-shaped precursor miRNA.
Pre-miRNAs are then transported into the cytoplasm by a
nuclear export factor and processed into a 19- to 25-
nucleotide double-stranded RNA. This duplex miRNA is
then incorporated into the RNA-induced silencing complex.
One strand remains in the complex and becomes the ma-
ture miRNA, whereas the other strand is often rapidly
degraded. The mature miRNA interacts with the target
mRNAs as a negative regulator; downregulating protein
expression [52] miRNAs has been implicated in the patho-
genesis of various diseases and has become an intriguing
target for therapeutic interventions [53].
Recently, the existence and functionality of miRNA path-
way components in human platelets has been reported [54].
Moving from these observations, in preliminary experi-
ments, our group has analyzed miRNA modulation in an
ex vivo model of ADP-induced platelet activation, founding
that platelets activation resulted in the expression of 435
miRNAs, 308 of which were modulated up to 2 h after
activation. These findings suggest, for the first time, a phys-
iological role for miRNAs in the regulation of platelet
function [55].
function could be modulated by targeting the previously
discussed ligands and/or receptors; this approach has
resulted in the development of several pharmacological
interventions.
ADP Receptors Antagonism
P2Y12 receptor antagonists include ticlopidine, clopidogrel,
prasugrel, ticagrelor, and other compounds under late-stage
development (cangrelor, elinogrel) [56]. Ticlopidine, clopi-
dogrel, and prasugrel are thienopyridines that selectively
and irreversibly inhibit the P2Y12 receptor. Other new re-
versible P2Y12 inhibitors are currently at different stages of
clinical development, including those which reversibly in-
hibit the P2Y12 receptor available for oral (such as ticagrelor
that has been recently approved for routine use in patients
with acute coronary syndrome [ACS]), intravenous
(cangrelor), or both oral and intravenous (elinogrel) routes
of administration.
TXA2 Pathway Antagonism
Antagonists of TP receptors may have different advantages
over aspirin as they not only block the effect of TXA2 on
platelets but also inhibit other ligands such as prostaglandin
(PG) endoperoxides and isoprostanes. Given the distribution
of TP receptors in platelets, in circulating inflammatory
cells, in the vascular wall, and in atherosclerotic plaques,
they also inhibit the effects of TXA2 over TP receptors on
vascular cells or in the plaque. A large trial is currently
comparing the efficacy and safety of terutroban versus as-
pirin in secondary prevention of cardiovascular events in
patients who have suffered a stroke or transient ischemic
attack (PERFORM Study) [57].

Therapeutic Modulation of Platelet Function Collagen Receptors Antagonism

In the last two decades, better understanding of platelets Collagen receptors antagonism is considered as a new prom-
biology induced researchers to investigate how platelet ising strategy for antithrombotic therapy. GPVI has emerged
304
as a powerful and safe pharmacological target preventing
thrombus formation without causing major bleeding com-
plications in experimental animal model. Injection of JAQ1,
a monoclonal antibody selective for GPVI, in mice results in
an acquired deficiency of the receptor that mimics a GPVI
knockout phenotype, causing a long-term antithrombotic
protection, despite a mild increase in bleeding times [58].
An alternative way of antagonism is the use of F(ab) frag-
ments of anti-GPVI that block the collagen-binding site of
the receptor. Also, this type of approach has resulted in
limited bleeding complications [59]. Other modalities of
antagonism are represented by competitive inhibition of
GPVI-binding sites on its substrate collagen through
GPVI-Fc fusion protein [60] or by the interference with
the GPVI/FcR gamma-chain intracellular signaling pathway
[61]. The favorable profile of GPVI antagonism derives
from a relatively minor role of GPVI in the physiological
hemostatic process, as opposed to a prime role in the for-
mation of in vivo (experimental) arterial thrombi following
injury of the arterial wall.
The collagenvWFGPIb complex, for its dominant role
in platelets adhesion, represents an alternative interesting
target for new antithrombotic drugs. Anti-vWF and anti-
GPIb antibody showed a potent antithrombotic effect in
vivo, without significant increase of bleeding risk [62],
while despite its unquestionable involvement in adhesion
process, contradictory results have been obtained in experi-
ments with integrin 21 antagonist [62]. More recently a
bivalent nanobody (ALX-0081) that binds with high affinity
to the A1 domain of vWF has been developed. It consists of
two identical building blocks bound to each other via a short
linker that stops the interactions between platelets and vas-
cular collagen. It selectively prevents thrombus formation
under high shear stress conditions [63]. In the proof of
concept phase 2 trial comparing ALX-0081 versus GPIIb/
IIIa inhibitor ReoPro in ACS patients, the nanobody showed
that 36 (19.9 %) patients reported bleeding events in the
ALX-0081 remedy group and 28 (15.3 %) patients reported
bleeding events in the ReoPro remedy group. Only three
ALX-0081-treated patients (1.7 %) and two ReoPro-treated
patients (1.1 %) showed a key bleeding event during the 30-
day period following the percutaneous coronary interven-
tion (PCI) process. However, ALX-0081 failed to meet the
primary endpoint of a 40 % reduction in total bleeding
events compared to the GPIIb/IIIa inhibitor ReoPro [64].
PAR Antagonism
Blocking exclusively thrombin-mediated platelet activation,
PAR modulation might act selectively on pathological
thrombosis. Two distinct PAR-1 antagonists, vorapaxar
and atopaxar, were investigated in phase 2 and phase 3
clinical studies. Vorapaxar is a high-affinity, orally active,
J. of Cardiovasc. Trans. Res. (2013) 6:299309
low-molecular-weight nonpeptide, competitive antagonist
of PAR-1. Despite preclinical and early clinical studies that
reported no significant increase in bleeding, the Thrombin
Receptor Antagonist for Clinical Event Reduction in ACS
(TRACER) trial, a phase 3 study designed to test the antith-
rombotic effect of vorapaxar administered in combination
with aspirin and/or clopidogrel, was stopped because of
increased intracranial bleeding in the vorapaxar group
[65]. These data have been also confirmed by the recently
published TRA2P trial, in which vorapaxar reduced the risk
of cardiovascular death or ischemic events in patients with
stable atherosclerosis but it was associated with an increas-
ing risk of moderate or severe bleeding, including intracra-
nial hemorrhage [66]. Atopaxar is a low-molecular-weight,
competitive antagonist of PAR-1 trialed for the treatment
and prevention of arterial thrombosis. In a small phase 2 trial
(Japanese-Lesson from Antagonizing the Cellular Effect of
Thrombin [J-LANCELOT]), atopaxar has been proved to be
generally well-tolerated, with no increase in major bleeding
[67]. These results presented atopaxar as a potential effec-
tive treatment of thrombosis and led to phase 3 trial for the
evaluation of therapeutic efficacy in the treatment and pre-
vention of cardiovascular disease [67]. Thus, in the near
future, PAR antagonism might represent a valid alternative
to the current antithrombotic therapy. Further studies are
needed to clarify its efficacy and safety profile.
GPIIb/IIIa Antagonism
Since the GPIIb/IIIa receptor is the final common pathway
that leads to platelet aggregation, direct inhibition of this
receptor is one of the therapeutic strategies widely used in
clinical practice [22]. This inhibition has been obtained by
using monoclonal antibody or blocking peptides. Presently,
abciximab, eptifibatide, and tirofiban are the GPIIb/IIIa
inhibitors most extensively studied in several randomized
trials involving thousands of patients with ACS or undergo-
ing PCI [23]. Abciximab is a large monoclonal antibody that
binds to the GPIIb/IIIa receptor as well as to the v3
integrin receptor of endothelial and smooth muscle cells
and to the m2 integrin found in leucocytes. Tirofiban
and eptifibatide are nonpeptide molecules with marked
specificity for the GPIIb/IIIa receptor. Thus, only abciximab
has the potential for direct inhibition of adhesion of platelets
and endothelial cells and of platelets and white cells, and as
a consequence, to inhibit or limit the inflammatory process
invariably present in ACS and after PCI. In randomized
placebo-controlled trials, the risk of recurrent ischemic com-
plications within 30 days after PCI was reduced by 4060 %
with abciximab and by 1535 % with eptifibatide or tirofi-
ban [23]. Moreover, abciximab has been associated with a
long-term decrease in mortality, an effect that cannot be
entirely attributed to the suppression of acute periprocedural
J. of Cardiovasc. Trans. Res. (2013) 6:299309
ischemic events, whereas mortality reduction has not been
observed to date with eptifibatide or tirofiban [24].
COX Pathway: Thrombosis, Inflammation, and Beyond
COX is the central enzyme of cascade reactions leading to
the conversion of AA to prostanoids, a cluster of bioactive
lipids known to affect multiple signaling pathways that
modulate physiological processes such as inflammation,
blood clotting, wound healing, blood vessel tone, and im-
mune responses. The first step of this pathway is the liber-
ation of AA from membrane phospholipid by phospholipase
A2. The free AA is converted via two independent enzymat-
ic reactions catalyzed by COX, first in PGG2 via a COX
function and then in PGH2 via a peroxidase function. PGH2
is then converted to other PGs, such as prostacyclins (PGI2)
and TXA2, by tissue-specific isomerases [68].
There are two different COX isoenzymes, known as COX-
1 and COX-2, that differ for regulatory mechanisms of ex-
pression, tissue distribution, substrate specificity, and prefer-
ential coupling to upstream and downstream enzymes [69].
Recently, an alternative splice variant of COX-1 has been
identified (COX-3). This variant is highly unlikely to be a
functional human protein because it has no COX activity [70].
COX-1 is a membrane-bound hemoglycoprotein of
71 kDa found in the endoplasmic reticulum of PG-
producing cells, constitutively expressed in most of tissue.
The enzyme structure is characterized by an EGF-like do-
main, a membrane-binding domain, and an enzymatic do-
main. The COX active site is a long hydrophobic channel that
is blocked by nonsteroidal anti-inflammatory drugs to prevent
the access of AA. COX-1-derived PGs are thought to mediate
physiological processes that require constant or rapid modula-
tion (or both), the so-called housekeeping functions, including
vascular homeostasis, gastrointestinal protection, and sodium
reabsorption in the kidney [71]. Furthermore, COX-1 is one of
the main controllers of platelet function, providing intermedi-
ate precursors for TXA2 synthesis.
Originally discovered as a protein upregulated during
inflammation and strongly decreased by glucocorticoids,
COX-2 is an inducible enzyme normally absent from tissues
(with the exception of kidney and parts of the brain, where
COX-2 is constitutive) that is mainly expressed by cells
involved in inflammation (e.g., macrophages, monocytes,
synoviocytes) in response to a wide range of extracellular
and intracellular stimuli (such as growth factors, tumor
promoters, or cytokines) [72]. Thus, it has emerged as the
isoform primarily responsible for the synthesis of prosta-
noids involved in acute and chronic inflammatory states
(i.e., PGE2). COX-2 structure closely resembles that of
COX-1; however, its active site has a slightly larger volume
and can accommodate specifically larger drugs [73].
305
COX-1 and COX-2 are differently regulated: (a) COX-2
requires considerably lower levels of hydroperoxides to
initiate COX catalysis than those required by COX-1; (b)
COX-2 activity occurs at lower levels of free AA than COX-
1 activity [11]; (c) COX-1 has the structural features of a
housekeeping gene, while COX-2 has the structural fea-
tures of an immediate early gene; and (d) both mRNA and
protein of COX-1 are more stable than those of COX-2 [74].
Pharmacological modulation of COX-1 activity can be
achieved by the use of aspirin, the oldest and most widely
prescribed antithrombotic drug. The antiplatelet effect has
been attributed to the irreversible acetylation of the serine
residue (Ser529) in platelet-expressed COX-1 that inhibits
the synthesis of PGH2 and, subsequently, the generation of
TXA2. The effect of aspirin on platelet TXA2 production
continues for the all lifetime of platelets, as consequence of
the aspirin ability to acetylate irreversibly COX-1 and the
inability of anucleated platelets to synthesize new COX-1
protein. A high level of inhibition of platelet COX-1 is
required in vivo to obtain antiplatelet efficacy (more than
95 % inhibition of TXA2 synthesis is required to observe
any clinical efficacy of aspirin). The inhibition of COX-2-
related pathologic manifestations (hyperalgesia and inflam-
mation) requires large doses of aspirin and a shorter dosing
interval (nucleated cells rapidly resynthesize the enzyme).
Novel Platelets Function: Link Between Inflammation
and Immunity
The central role of platelets in hemostasis and thrombosis is
well-known; however, platelets probably contribute to several
other processes, such as inflammation and immunity. This
concept took a long time to get consensus in the scientific
community. In addition to their traditional contribution in clot
formation, platelets are also specialized in further activities
and intercellular interactions that make them key effectors in
inflammation and in the continuum of innate and adaptive
immunity [75]. For example, they can recruit leukocytes and
progenitor cells to sites of vascular injury and inflammation
[75]; they release proinflammatory, anti-inflammatory, and
angiogenic factors and microparticles into the circulation
[76], and they spur thrombin generation [74].
Platelets also express immunoreceptors on their surfa-
ces, including Fc receptors that recognize immunoglobulins
of the IgG, IgE, and IgA classes. Like platelet integrins,
these immunoreceptors seem to influence adhesion and,
more importantly, inflammatory and immune responses
[77]. Via these receptors, platelets may contribute to innate
and adaptive immunity. As part of innate immunity, they
possess, in fact, rudimentary antibacterial and phagocytic
activity by which platelets interact with bacteria, viruses,
and parasites. This interaction induces platelet activation
306
and secretion of antimicrobial peptides. Furthermore, in
platelet granules are stored many proinflammatory cyto-
kines (e.g., IL-1), which modulate the inflammatory/im-
mune responses [77]. More interestingly, platelets express
several functional Toll-like receptors (TLRs), such as TLR-
2, TLR-4, and TLR-9. It has been shown that the interaction
between platelets TLR-4 and lipopolysaccharide from the
gram-negative bacteria activates platelets and induces plate-
letneutrophil cross-talk, leading to neutrophil degranula-
tion and release of extracellular traps that can kill the
bacteria [77].
Platelets are also actively involved in adaptive immunity.
They contain functional transforming growth factor beta
(TGF-), which is essential for the development of naturally
occurring regulatory T cells (nTreg) or T helper 17 cells
(Th17), depending on the local cytokine environment [78].
This involvement seems to be mediated by P-selectin
(CD62P), which plays an important role in the development
of the Th1 immune response [79], and CD40L, which plays an
important role in supporting immunoglobulin class switch and
augmenting CD8+ T cell function during viral infection [80].
Platelet activation induces P-selectin translocation from
the -granule to the cell surface. P-selectin can then interact
Fig. 2 Platelets as bridge between hemostasis and immunity. Throm-
bin activation of PARs, in concert with platelet-derived CD154, sero-
tonin (5HT), platelet factor 4 (PF4), and regulated and normal T cell
expressed and secreted (RANTES), has now been discovered to opti-
mize the process of antigen presentation to initiate and steer the
phenotype of subsequent adaptive immune responses. Platelets express
CD154, which can be secreted by TLR induction or remotely delivered
by platelet-derived microvesicles to augment both dendritic and B cell
J. of Cardiovasc. Trans. Res. (2013) 6:299309
with peripheral lymph nodes addressin on high endothelial
venules (HEV) and P-selectin glycoprotein ligand-1 (PSGL-
1) on lymphocytes simultaneously, thus mediating the roll-
ing and recruitment of lymphocytes to HEV of peripheral
lymph nodes. Interestingly, platelets also contain multiple
anti-inflammatory cytokines, such as TGF- [81], and it has
been reported that metastasizing tumor cells induce platelets
to secrete TGF-, which inhibits the antitumor activity of
natural killer cells. Furthermore, platelets contain a large
amount of TSP-1, which comprises around 25 % of the total
protein content of platelet -granules [82]. It has been
demonstrated that TSP-1 not only activates the anti-
inflammatory cytokine TGF-1 [83] but also inhibits the
phagocytic capacity of macrophages. In summary, platelets
participate not only in proinflammatory responses but also
they might modulate that balance between inflammation and
immune responses (Fig. 2).
Conclusions
During the last decade, important advances in our under-
standing of the pathophysiology of platelets have been
activation. In a similar capacity, 5HT can activate T cells and induce
autocrine production of 5HT to amplify this process. PF4 directly
interacts with CXCR3 to increase T cell recruitment or forms dimers
with RANTES that recruits monocytes and T cells. The activation of
PAR on dendritic cells enhanced the production of proinflammatory
and prothrombotic mediators, a potential feedback system to amplify
antigen presentation
J. of Cardiovasc. Trans. Res. (2013) 6:299309
made. These small cell fragments play a critical role in all
stages of the complex, dynamic process of hemostasis and
thrombus formation. Their function is the result of the
interaction between different agonists and specific receptors
at platelet surface, triggering intracellular signaling that
leads to platelet activation; this is accompanied by the
release of different factors and inflammatory substances.
These mediators act in an orchestrated manner with the
vascular endothelium and other blood cells and coagulation
factors to promote not only the growth of the platelet plug
by recruiting new platelets but also to consolidate the throm-
bus by means of post-aggregation signaling events, clot
retraction, and controlled formation of a fibrin network.
On the other hand, compensatory molecules and signaling
events also starts to limit undesirable platelet accumulation
and pathological thrombosis. Based on new evidence, pla-
telets might be a bridge between different extracellular
functions, beyond the coagulative cascade, thus the im-
provement of our knowledge on these extracoagulative
actions may also result in the development of novel thera-
peutic approaches.
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