ANTIPLATELET THERAPY

The Role of Platelets in Atherothrombosis

Zane S. Kaplan1 and Shaun P. Jackson1

1Australian
Centre for Blood Diseases, Alfred Medical Research and Education Precinct, Monash University,
Melbourne, Australia

Platelets have evolved highly specialized adhesion mechanisms that enable cell-matrix and cell-cell interactions
throughout the entire vasculature irrespective of the prevailing hemodynamic conditions. This unique property of
platelets is critical for their ability to arrest bleeding and promote vessel repair. Platelet adhesion under conditions of
high shear stress, as occurs in stenotic atherosclerotic arteries, is central to the development of arterial thrombosis;
therefore, precise control of platelet adhesion must occur to maintain blood fluidity and to prevent thrombotic or
hemorrhagic complications. Whereas the central role of platelets in hemostasis and thrombosis has long been
recognized and well defined, there is now a major body of evidence supporting an important proinflammatory function
for platelets that is linked to host defense and a variety of autoimmune and inflammatory diseases. In the context of the
vasculature, experimental evidence indicates that the proinflammatory function of platelets can regulate various
aspects of the atherosclerotic process, including its initiation and propagation. The mechanisms underlying the
proatherogenic function of platelets are increasingly well defined and involve specific adhesive interactions between
platelets and endothelial cells at atherosclerotic-prone sites, leading to the enhanced recruitment and activation of
leukocytes. Through the release of chemokines, proinflammatory molecules, and other biological response modula-
tors, the interaction among platelets, endothelial cells, and leukocytes establishes a localized inflammatory response
that accelerates atherosclerosis. These inflammatory processes typically occur in regions of the vasculature
experiencing low shear and perturbed blood flow, a permissive environment for leukocyte-platelet and leukocyte-
endothelial interactions. Therefore, the concept has emerged that platelets are a central element of the atherothrom-
botic process and that future therapeutic strategies to combat this disease need to take into consideration both the
prothrombotic and proinflammatory function of platelets.

Introduction leukocyte interaction. The importance of local blood flow distur-

Platelets are anuclear cell fragments derived from the cytoplasm of
BM megakaryocytes1 that circulate in the bloodstream of humans
for 7-10 days. The primary hemostatic function of platelets has been
recognized for more than a century, and the molecular processes
regulating the adhesive function of platelets have been elucidated in
great detail.2-4 In addition to preventing excess bleeding, platelets
also play an important role in surveying and maintaining the
integrity of the endothelium, in part through the release of proangio-
genic cytokines and growth factors. Dysregulated platelet-
endothelial interactions have increasingly been recognized as an
important pathogenic mechanism in the development of atheroscle-
rosis. In response to specific proinflammatory signals, endothelial
cells become more adhesive toward platelets, stimulating the
production of various platelet-derived inflammatory molecules that
provide a positive feedback loop for further endothelial cell
activation. Endothelial-bound platelets are highly effective at recruit-
ing leukocytes from flowing blood and also enhance leukocyte
adhesion and transmigration to the site of the proinflammatory
stimulus. Therefore, pathological derangement of these key interac-
tions among platelets, endothelial cells, and leukocytes facilitates
the inflammatory process that underlies the development of
atherosclerosis.
This review outlines the key pathways used by platelets to facilitate
the development of vascular inflammatory lesions linked to the
development of atherosclerosis, with a focus on the mechanisms
leading to dysregulation of the platelet-endothelium and platelet-
bances in regulating the atherothrombotic process is briefly dis-
cussed. A brief overview of the well-defined platelet adhesion
mechanisms regulating platelet-vessel wall and platelet-platelet
interactions and a discussion of recent advances in the understand-
ing of the platelet procoagulant response are presented first.
Hemostatic and prothrombotic function of platelets
Under normal physiological conditions, platelets circulate in close
proximity to the endothelium without forming stable adhesion
contacts due to the anti-adhesive properties of quiescent endothelial
cells. However, after vascular injury, platelets rapidly adhere to sites
of endothelial disruption to establish a hemostatic plug that prevents
excess blood loss. This process is critically dependent on the
efficiency of platelet adhesion to the subendothelial matrix, as well
as on the ability of the platelets to undergo rapid biochemical and
morphological changes that support aggregation and the localized
activation of the coagulation cascade.
Platelet recruitment (Figure 1)
The mechanisms supporting platelet adhesion to the damaged vessel
wall have been thoroughly investigated and well defined, so only a
brief description will be provided here. After damage to the vessel
wall, subendothelial matrix proteins that are highly reactive to
platelets, including collagen, VWF, fibronectin, and laminin, be-
come exposed to the blood and immediately engage specific
receptors on the platelet surface. The contribution of specific ligands
and receptors mediating platelet adhesion is critically dependent on

Hematology 2011 51
Figure 1. Hemostatic and prothrombotic function of platelets under flow. (A) Platelet adhesion mechanisms operating under arterial flow. After
disruption of the endothelium, platelets are rapidly recruited from flowing blood via a tethering mechanism dependent on the interaction of platelet
GPIb-V-IX and VWF. This adhesive bond has a rapid dissociation rate, resulting in platelet translocation on the vessel wall. (B) Platelet firm adhesion
and aggregation. Translocating platelets engage collagen in the vessel wall through their adhesion receptors GPVI and 21. GPVI is the major
collagen receptor inducing intracellular calcium flux necessary for stable platelet adhesion, cytoskeletal reorganization, IIb3 activation, and the release
of the soluble agonists (ADP and TxA2). These agonists act through specific G-protein coupled receptors to amplify the platelet activation response.
TxA2 is derived from the metabolism of arachidonic acid via the COX-1 pathway, whereas ADP is released from platelet-dense granules. Locally
generated thrombin also enhances platelet activation through proteolytic cleavage and activation of PARs. Activated platelets form stable aggregates
through IIb3 engagement of VWF and fibrinogen. These molecular pathways of amplification of platelet activation are the target of both clinically
available and experimental therapeutic interventions in the treatment of atherothrombotic disease. (C) Stabilization of the platelet thrombus. The primary
hemostatic plug is consolidated by fibrin generation at the site of injury and throughout the developing thrombus. -Thrombin generation at the injured
vessel wall is critically dependent on TF, whereas thrombin generation on the surface of activated platelets is likely to evolve both TF and contact
factor dependent activation of blood coagulation. UHF indicates unfractionated heparin; LMWH, low-molecular-weight heparin; VKA, vitamin K
antagonist; PGH2, prostaglandin H2; AA, arachidonic acid, PLA2, phospholipase A2; IP3R, inositol trisphosphate receptor; ITAM, immunoreceptor
tyrosine-based activation motif; FcR, Fc receptor gamma.

the prevailing blood-flow conditions. Under conditions of high
shear stress, as encountered in arterioles and stenotic arteries, VWF
plays a critical role in recruiting platelets from blood flow.5 This
tethering function of subendothelial VWF is dependent on the
interaction of its A1 domain with the glycoprotein Ib (GPIb)
component of the platelet GPIb-V-IX complex.6 Circulating plasma
VWF has limited binding potential for GPIb, however, once
immobilized onto subendothelial collagen (type I, III, and VI), the
unfolded VWF macromolecule provides a linear array of A1
domains that facilitate binding to multiple GPIb receptors.7,8 The

52 American Society of Hematology

bond between GPIb and VWF has a rapid dissociation rate that is
unable to support stable adhesion, requiring the contribution of
other ligand-receptor interactions to promote stable adhesion.9 The
fundamental importance of both VWF and GPIb for the adhesive
function of platelets is underscored by the severe bleeding disorder
experienced by patients with qualitative or quantitative defects in
these molecules.10
Firm adhesion and activation (Figure 1)
Once tethered to the site of vascular injury, platelets form stable
adhesion contacts with collagen and other matrix macromolecules.
Platelet binding to collagen is mediated by GPVI11,12 and integrin
21,13 whereas fibronectin and laminin engage integrin 51 and and leukocytes. TF expression and activation helps to stabilize the
61, respectively.4 Isolated deficiency of any of these receptors
does not cause a severe bleeding disorder, suggesting considerable
redundancy in the mechanisms regulating platelet-vessel wall
interactions. Once adherent, platelets undergo a remarkably com-
plex series of morphological and biochemical changes that lead to
the release of platelet granule contents and up-regulation of the
adhesive function of integrin IIb3. Activated IIb3 binds multiple suggested a potentially important role for the contact phase of blood
ligands, including VWF,14,15 fibrinogen,16 fibrin and fibronectin,17
and is indispensable for the formation of stable platelet aggregates.18
Therefore, similar to GPIb, quantitative or qualitative defects in
IIb3 leads to a major defect in the hemostatic function of platelets
and a severe bleeding disorder (Glanzmann thrombasthenia).19
Integrin IIb3 deficiency also provides protection against arterial
thrombosis, thus pharmacological IIb3 antagonism represents a
highly effective antithrombotic approach.20,21
Soluble agonists amplifying platelet activation (Figure 1)
Central to platelet activation is the generation and release of soluble
agonists at sites of vascular injury. These include thromboxane A2
(TxA2) which is synthesized from arachidonic acid via cyclooxygen-
ase (COX) and thromboxane synthase, and ADP released from
platelet dense granules. These endogenous agonists can act in an
autocrine or paracrine manner to enhance platelet activation by
engaging specific G protein coupled receptors; P2Y122 and P2Y12 23
(ADP) and the thromboxane receptors TP and TP.24 ADP and
TxA2 act in a cooperative manner to enhance platelet activation and
thrombus formation, thus pharmacological targeting of TxA225
generation and/or the P2Y1226 receptor are effective strategies to
reduce thrombus propagation at sites of vascular injury.27,28
Thrombin (Figure 1)
In addition to the synthesis and release of soluble agonists, platelets
provide a catalytic surface for the assembly of coagulation com-
plexes necessary for -thrombin generation. Thrombin is among the
most potent stimulators of platelets through proteolytic cleavage
and activation of platelet protease-activated receptors (PARs),
specifically PAR1 and PAR4, on human platelets.29 Also central to
the hemostatic function of thrombin is its ability to generate fibrin
polymers, a key step stabilizing formed thrombi. Therefore, pharma-
cological inhibitors of thrombin (eg, direct thrombin inhibitors),
upstream coagulation proteases (vitamin K antagonists and factor
Xa [FXa] inhibitors), and, potentially, thrombin receptors (PAR1
antagonists), represent effective strategies to limit arterial thrombus
growth.27
Procoagulant function of platelets: emerging
concepts
Current therapeutic strategies aimed at reducing thrombins effects
in vivo primarily target various components of the coagulation
Hematology 2011
cascade, thus affecting thrombin generation at the vessel wall as
well as within the body of thrombus. However, recent progress in
understanding the mechanisms by which platelets support blood
coagulation have raised the possibility that selectively inhibiting
platelet procoagulant function may provide a targeted approach to
specifically reduce thrombin generation within the thrombus.
Platelet regulation of the contact phase of blood
coagulation (Figure 2)
The generation of thrombin at the injured vessel wall is contingent
upon the expression of tissue factor (TF) on the surface of
fibroblasts, smooth muscle cells, and potentially endothelial cells
active conformation of FVIIa,30 triggering the extrinsic pathway of
blood coagulation. The mechanisms underlying the generation of
thrombin within the body of a developing thrombus remains
controversial. Whereas TF is expressed on the surface of activated
leukocytes and microparticles and has been demonstrated to be
incorporated within the thrombus, recent experimental evidence has
coagulation in promoting arterial thrombus growth. This rekindled
interest in the role of contact factors (eg, FXII, FXI, and high-
molecular-weight kininogen) in promoting blood coagulation in
vivo stemmed from the observation that FXII-deficient mice are
protected from vasoocclusive thrombi in arterial thrombosis mod-
els.31 Similarly, FXI deficiency32,33 and pharmacological antago-
nism34 of the contact phase of coagulation leads to a similar defect in
arterial thrombosis. Given that humans with complete FXII defi-
ciency do not have a significant hemostatic defect, this has raised the
possibility that therapeutic targeting of this process may lead to the
development of safer antithrombotics. How platelets activate the
contact phase of coagulation during thrombus development has
remained a vexing issue.35 Recently, Muller et al identified inor-
ganic polyphosphate released from activated platelets as a major
regulator of FXII activation.36 Pharmacologic exploitation of this
pathway may enable targeting of platelet polyphosphate by phospha-
tases to reduce thrombotic risk while leaving hemostatic responses
intact.
Identification of the elusive phospholipid scramblase
The contribution of platelet-derived phospholipids to thrombin
generation has long been recognized; however, the biological
mechanisms regulating this procoagulant platelet function have
remained poorly defined. In resting platelets, membrane phospholip-
ids are asymmetrically distributed: phosphatidylcholine and sphin-
gomyelin are primarily localized in the outer leaflet of the plasma
membrane, whereas phosphatidylethanolamine and phosphatidylser-
ine (PS) are largely confined to the inner leaflet. This asymmetry is
maintained through the action of two membrane lipid transporters,
flippase and floppase.37,38 After platelet stimulation by potent
agonists, this asymmetry is lost due to activation of Ca2-dependent
phospholipid scramblases that bidirectionally transport phospholip-
ids.39 Scramblase activity therefore results in the expression of
negatively charged phospholipids, especially PS, on the outer leaflet
of the platelet membrane, thus providing the requisite surface for
assembly of coagulation factors required for thrombin generation
(Figure 2). Defects in platelet PS expression are rare; however, in
affected individuals, this leads to a major hemostatic disturbance, as
described in Scott syndrome.40 Until recently, the identity of the
phospholipid scramblase had remained elusive, but recent studies
have identified a plasma membrane protein transmembrane protein
16F (TMEM16F) that confers Ca2-dependent scrambling of phos-
pholipids. A mutation in the TMEM16F gene resulting in premature
53
Figure 2. Procoagulant function of platelets. (A) Plasma membrane phospholipids are asymmetrically distributed in resting platelets, with PS
confined to the inner leaflet. After platelet activation by potent agonists, high levels of sustained intracellular calcium triggers scramblase activity.
Scramblase activation results in the loss of phospholipid asymmetry and PS exposure on the outer leaflet of the platelet membrane, providing the
requisite surface for rapid thrombin generation. (B) PS exposure can be induced through two distinct cell-death pathways; apoptosis and necrosis.
Apoptosis results in assembly of the Bak/Bax mitochondrial membrane pore, causing release of cytochrome C (CytC), caspase activation, and
substrate proteolysis. Necrotic cell death can be initiated by elevated cytosolic Ca2, causing a loss in mitochondrial membrane potential and
subsequent formation of a mitochondrial permeability transition pore (mPTP). mPTP formation results in bioenergetic failure through ATP depletion with
consequent reactive oxygen species (ROS) generation, leading to loss of membrane integrity. (C) Platelets can also contribute to thrombin generation
via activation of FXII through the release of polyphosphate from dense granules. (D) Whether there are additional cell death pathways regulating PS
exposure and the procoagulant function of platelets remains unclear. PC indicates phosphatidylcholine.

termination of the protein has been demonstrated in a patient with
Scott syndrome.41
Cell death and the procoagulant function of platelets
(Figure 2)
Procoagulant platelets expressing PS exhibit distinct morphologic
and biochemical features relative to fully activated platelets. Many
of these changes are indicative of dead or dying cells, and include
membrane blebbing, microvesiculation, activation of intracellular
proteases, and the cleavage of cytoskeletal proteins, PS surface
exposure, and alterations in mitochondrial membrane potential.42
These results suggest that the pathways regulating cell death in
platelets may play an important role in regulating the platelet
procoagulant phenotype. Programmed cell death is orchestrated by
several pathways, including apoptosis, necrosis, and autophagy.
Apoptosis is an energy-dependent process contingent upon caspase
activation and is critical for the removal of senescent cells. Necrosis
is typified by bioenergetic failure of the cell resulting in membrane
instability and the release of intracellular contents into the extracel-
lular environment. Recently, evidence has emerged that platelet
stimulation by potent agonists initiates a tightly regulated cell death
pathway mediated by sustained high levels of cytosolic Ca2,
culminating in distinct morphological and biochemical changes that
are consistent with a necrotic cell death pathway.43 Direct induction
of apoptosis can also induce PS expression and platelet procoagu-
lant function,43 raising the possibility that multiple cell death
pathways may regulate thrombin generation on the surface of
platelets. In addition to promoting thrombin generation, procoagu-
lant platelets may also contribute to the inflammatory response by
generating high levels of the proinflammatory lipid platelet-
activating factor (PAF) and by enhancing leukocyte adhesion.44
Proinflammatory role of platelets
Promiscuous platelets
The growing list of pathophysiological processes in which platelets
have a proposed role is in part a reflection of the large number of

54 American Society of Hematology

Figure 3. Platelet-endothelial cell interactions. (A) The healthy endothelium. The anti-adhesive phenotype of endothelial cells is maintained through
3 intrinsic pathways: the ecto-ADPase/CD39/NTPDase pathway, which metabolizes ADP, and the PGI2 and NO pathways, which inhibit platelet
activation through the stimulation of cAMP and cGMP production, respectively. (B) Endothelial activation in hyperlipemia. In a hyperlipidemic milieu, the
endothelium becomes inflamed as a result of modified lipoprotein particles and reactive oxygen species (ROS) accumulating in the intima. This leads to
the expression of adhesive ligands (VWF and P- and E-selectins) on the endothelium. (C) Platelet adhesion to the inflamed endothelium and platelet
endothelial cross-talk. The expression of VWF and P-selectin on the endothelial surface supports platelet tethering and rolling. Subsequently, stable
platelet adhesion occurs through fibrinogen-IIb3 complexes binding to v3 or ICAM-1 on endothelial cells. Adherent platelets secrete numerous
bioactive substances that alter the chemotactic and adhesive properties of the endothelial cells. Platelet-derived IL-1 induces endothelial secretion of
IL-6 and IL-8 and surface expression of ICAM-1, v3, and MCP-1. Platelet CD40 ligand binds CD40 on endothelial cells, resulting in up-regulation of
adhesive molecules (ICAM-1, VCAM-1, and E- and P-selectin), cytokine and TF release, and reduction in NO synthesis. Ecto ADPase indicates
ecto-adenosine diphosphatase; NTPDase, nucleoside triphosphate diphosphohydrolase.

different cell types with which platelets can interact. These include
endothelial cells, neutrophils, monocytes, dendritic cells, cytotoxic
T-lymphocytes, malaria-infected red blood cells, and various tumor
cells. In the context of atherosclerosis, the interaction of platelets
with endothelial cells and leukocytes appears to be important for the
initiation and propagation of inflammatory processes in the arterial
wall.45
for P-selectin include GPIb and P-selectin glycoprotein-1 (PSGL-
1),51,52 which support tethering and rolling, but not firm adhesion on
inflamed endothelium. Whereas there is some uncertainty as to the
exact mechanism supporting stable platelet adhesion to activated
endothelium, in vitro and in vivo models suggest that binding of
IIb3 to fibrinogen bound to endothelial v3 or ICAM-1 facilitates
firm platelet adhesion and activation.53,54

Platelet-endothelial cell adhesive interactions (Figure 3) Chemical signals

While platelets continuously circulate in close proximity to the
endothelium, under physiological conditions, they typically do not
form sustained adhesive interactions. The anti-adhesive phenotype
of endothelial cells is controlled by the three intrinsic pathways; the
nitric oxide (NO) pathway, the ecto-ADPase/CD39/NTPDase path-
way, and the ecosanoid-arachidonic acid-prostacyclin (PGI2) path-
way.46 It is increasingly recognized that inflammatory stimuli
facilitate sustained endothelial-platelet adhesion by perturbing these
anti-adhesive mechanisms and increasing surface expression of
endothelial adhesion molecules.47 The adhesion of platelets to
inflamed endothelium involves a similar coordinated multistep
process as occurs in hemostasis and thrombosis, including platelet
tethering, surface translocation, and firm adhesion.48 Initiation of the
inflammatory insult to the endothelium in atherosclerosis is partly
due to the accumulation of modified oxidized lipoprotein particles
in the setting of hyperlipidemia.49 This leads to surface expression
of endothelial selectins, including P- and E-selectin, as well as
membrane expression of VWF.48,50 The platelet counterreceptors
Platelets activated through their interactions with inflamed endothe-
lium release a vast array of inflammatory and mitogenic mediators
(Table 1) into the local microenvironment.55 These platelet-derived
bioactive substances alter the chemotactic and adhesive properties
of the endothelial cells, enabling monocytes and other leukocytes to
adhere and transmigrate through the endothelium to sites of
inflammation.48,56 In vivo models of atherogenesis suggest that
these interactions between endothelial cells and activated platelets
are likely to be critical in the initiation of atherosclerosis.57,58
IL-I (Figure 3). IL-I is a platelet derived cytokine that has been
postulated to be a principal mediator of platelet-induced endothelial
activation.59,60 It is not stored in platelet granules, but is believed to
be synthesized by platelets from constitutional prepackaged mRNA
several hours after thrombin stimulation or integrin-mediated adhe-
sion.61 IL-I stimulation of endothelial cells induces secretion of
IL-6 and IL-8 and significantly increases the endothelial surface
expression of ICAM-1, v3, and monocyte chemotactic protein-1

Hematology 2011 55
Table 1. Bioactive platelet mediators underlying atherothrombosis
Localization within the
platelet Released molecule Target cell Function
Dense granules ADP Platelet P2Y1 and P2Y12 Platelet activation
ATP Platelet P2X1 Platelet activation and sensitization to other
agonists
5HT Platelet 5HT2A Weak platelet agonist
Endothelium 5HT2B NO synthesis: vasodilation, platelet inhibitor
Vascular smooth muscle Vasoconstriction
Monocytes T-cell activation: proinflammatory
granules
Adhesion receptors IIb3 Platelet Platelet aggregation, platelet-endothelial, and
platelet-leukocyte adhesion
Coagulation factors FV Procoagulant
Adhesive ligands VWF Platelet Platelet-endothelium and platelet-vessel wall
tethering and platelet aggregation
Fibrinogen Platelet Platelet aggregation, fibrin clot formation, and
platelet-leukocyte adhesion
Fibronectin Platelet Platelet-endothelial and platelet-leukocyte
adhesion
P-selectin Platelets, endothelial cell and Adhesion of leukocytes to platelets and
leukocytesPSGL-1 endothelial cells
Chemokines CXCL4 (PF-4) Leukocytes Synergizes with other cytokines to promote
leukocyte adhesion and monocyte
differentiation
CXCL7 (PBP, -TG, CTAP-III, Leukocytes Enhances neutrophil and monocyte adhesion
and NAP-2) and neutrophil transendothelial migration
CCL5 (RANTES) Endothelium/leukocytes Promotes monocytes adhesion to the
endothelium and chemokine synthesis
Growth factors PDGF Vascular smooth muscle Stimulates vascular smooth muscle cell
cells migration and proliferation associated with
intimal hyperplasia
Synthesized cytokines IL-1 Endothelial cells Up-regulation of leukocyte adhesion molecules
and stimulation of endothelial release of
proinflammatory cytokines
granule and platelet
membrane
CD40 Ligand Endothelial cell CD40 Upregulation of leukocyte adhesion molecules
and stimulation of endothelial release of
proinflammatory cytokines
Proinflammatory lipids TxA2 Platelets Platelet activation
Leukotriene B4 Leukocytes (BLT-1 and Chemo-attractant and promotion of monocyte
BLT-2) differentiation into foam cells and neutrophil
activation
PAF Platelet/leukocytes Platelet agonist and promotes neutrophil
adhesion

(MCP-1).48,60,62 ICAM-1 and MCP-1 up-regulation by endothelial
cells in response to IL-I is dependent on the activation of NF-B.63
Through this potential mechanism, platelets are able to significantly
enhance monocyte and neutrophil adhesion to the endothelium.
CD40 ligand (CD40L-CD154; Figure 3). CD40 ligand is an
important platelet-derived bioactive mediator of endothelial cell
activation. CD40L is a transmembrane protein with structural
homology to TNF-.64 CD40L was initially identified on the surface
of activated CD4 T cells and is central to the function of the
humoral immune system by supporting B-cell differentiation and
immunoglobulin class switching.65 It was subsequently demon-
strated that platelets are a rich source of CD40L,66 with 95% of
circulating CD40L being of platelet origin.67 After release from
platelets, CD40L interacts which CD40 expressed on numerous cell
types, including endothelial cells, smooth muscle cells, leukocytes,
fibroblasts, and platelets.64,68 In endothelial cells, CD40L binding
with its cognate receptor CD40 results in up-regulation of ICAM-1,
VCAM-1, and E- and P-,selectin as well as the release of IL-6 and
TF.69 CD40L inhibits endothelial NO production by destabilizing
NO synthase mRNA64,70; therefore, CD40L binding to endothelial
cells results in a proatherogenic and proinflammatory phenotype.64
Platelet-leukocyte cross-talk in atherogenesis
The central role of leukocytes, especially monocytes and T lympho-
cytes, in the development of atherosclerosis is well established.71
There is increasing evidence that activated platelets adherent to the
inflamed endothelium may enhance leukocyte recruitment, activa-
tion, and transmigration, thereby enhancing the inflammatory
processes underlying atherosclerosis.
Leukocyte recruitment to endothelial-bound platelets is a coordi-
nated, multistep process involving selectin-mediated tethering and
rolling, followed by 2 integrinmediated stable adhesion (Figure

56 American Society of Hematology

Figure 4. Platelet-leukocyte interactions promoting atherogenesis. (A) Platelet-mediated leukocyte recruitment and adhesion. Leukocyte
recruitment to endothelial-bound platelets occurs in a multistep coordinated process. Initial tethering of leukocytes is mediated by the interaction of
P-selectin expressed on the platelet surface with its cognate receptor leukocyte PSGL-1. Ligation of PSGL-1 promotes activation of leukocyte 2
integrins (Mac-1 and LFA-1), necessary for stable leukocyte adhesion. Note that Mac-1 can engage several platelet ligands, including GPIb, ICAM-2,
and JAM-3, as well as fibrinogen bound to IIb3. Release of the chemokine RANTES at sites of atherogenesis leads to enhanced monocyte adhesion.
Monocytes subsequently migrate through the endothelium and differentiate into macrophages, which engulf lipids and transform into foam cells.
(B) Platelet-leukocyte cross-talk. Bioactive mediators derived from -granules of activated platelets, including PF4, synergize with RANTES to enhance
leukocyte adhesion and monocyte differentiation. RANTES, acting in concert with P-selectin, results in MCP-1 and IL-8 secretion by monocytes.
CTAP-III is another chemokine released from the platelet -granules, which is subsequently converted into active NAP-2 by the neutrophil membrane
associated serine-protease cathepsin G. NAP-2 enhances leukocyte adhesion and neutrophil transendothelial migration. Up-regulation of COX-2 in
monocytes via pathways involving IL-I and P-selectin results in increased production of proinflammatory mediators, including PAF, leukotrienes, and
TxA2 via transcellular eicosanoid metabolism. P-sel indicates P-selectin; AA, arachidonic acid.

4). Platelet P-selectin has a central role in mediating the initial
capture and rolling of leukocytes through interactions with its key
counterreceptor P-selectin glycoprotein 1 (PSGL-1).51,72 Ligation of
PSGL-1 induces signaling events culminating in the activation of
the leukocyte 2 integrins, including macrophage antigen-1 (Mac-1,
M2) and lymphocyte function-associated antigen-1 (LFA-1, L2),
which are required to mediate stable leukocyte adhesion. Mac-1 is
the principal 2 integrin that facilitates firm adhesion onto the
surface of platelets via its binding to multiple ligands, including
GPIb,73 ICAM-2,74 and junctional adhesion molecule-3 (JAM-
3),75 as well as through fibrinogen bound to IIb3.48,76
Platelet activation with consequent degranulation results in the
liberation of numerous chemokines stored in platelet -granules.
These chemokines can either be released into the circulation or
displayed on the platelet surface, where they exert numerous
biological activities instrumental in atherosclerosis.77,78
CXC chemokines NAP-2 and PF4 (Figure 4)
The two most abundant CXC chemokines stored in platelet
granules are CXCL7 and platelet factor 4 (PF4). CXCL7 has
several molecular variants, including the inactive precursor, connec-
tive tissue-activating peptide III (CTAP-III), which is enzymatically
converted into active neutrophil activating protein-2 (NAP-2)
through proteolysis by the neutrophil membrane-associated serine-
protease cathepsin G.79 NAP-2 has been demonstrated to induce
neutrophil and monocyte adhesion in vitro and neutrophil transendo-
thelial migration.78,80 The chemotactic properties of PF4 on its own
are controversial because it does not exert classical chemokine

Hematology 2011 57
functions at submicromolar concentrations.78,81 However, PF4 act-
ing in concert with other chemokines such as the platelet-derived
CC chemokine regulated upon activation normal T-cell expressed
and secreted (RANTES) may enhance leukocyte adhesion and
promote monocyte differentiation and atherosclerosis. These PF4-
RANTES heterodimers have been proposed to represent potential
therapeutic targets in the treatment of atherosclerosis.82
CC chemokine RANTES (Figure 4)
The proinflammatory CC chemokine RANTES is released from
activated platelets and deposited on activated endothelium, where it
is able to promote adhesion of monocytes.83 A prerequisite for the in
vivo activity of RANTES function is for it to bind to glycosamino-
glycans and to form higher-order oligomers.84 Further, through its
interactions with other modulators, including MCP-4, PF4, and
P-selectin, RANTES has been implicated in inflammation, athero-
genesis, and vascular remodeling after injury. Interestingly, P-
selectin engagement with PSGL-1 on monocytes is insufficient for
the induction of MCP-1, requiring the cooperation of platelet-
derived RANTES for chemokine synthesis by monocytes.85 Further,
monocytes costimulated by RANTES and P-selectin secrete a
different cytokine profile, including MCP-1 and IL-8.85 Platelet
activation results in the release of microparticles that are able to
facilitate the deposition of RANTES on the inflamed and atheroscle-
rotic endothelium, thus promoting monocyte adhesion.86
Proinflammatory lipids (Figure 4)
In addition to being storage houses for cytokines and chemokines,
platelets also generate lipid-derived inflammatory mediators. The
adhesion of platelets to leukocytes and the endothelium results in
transcellular metabolism of eicosanoids, leading to the local synthe-
sis of proinflammatory lipids such as PAF, leukotrienes, and
TxA2.87 The imbalance of inflammatory mediators is further exacer-
bated by monocyte up-regulation of COX-2 expression via interac-
tions involving P-selectin derived from activated platelets and
IL-I. This up-regulation of COX-2 enables further synthesis of
proinflammatory eicosanoids.88 PAF is a potent platelet agonist and
inflammatory mediator and has also been shown to regulate firm
neutrophil adhesion on the surface of immobilized spread platelets.44
Atherothrombosis and disturbed blood flow
An important determinant influencing platelet-endothelial and plate-
let-leukocyte interactions at atherosclerotic-prone sites is the local
blood flow conditions. It is well established that atherosclerotic
lesions develop at arterial branch points, which are typically areas of
low shear and disturbed flow. Altered flow has a significant effect on
the adhesive properties of platelets and leukocytes, as well on the
morphology and function of endothelial cells. Rheological factors
also contribute to the deposition of lipids and other modulators of
atherosclerosis.
Throughout much of the vasculature, blood flow is laminar, with
adjacent fluid layers traveling parallel to each other but at different
velocities because of the fluid drag exerted by the vessel wall. This
phenomenon leads to the generation of shear forces between
adjacent fluid planes. These flow profiles apply to straight, healthy
vesselsat arterial branch points, curvatures, and areas of stenosis,
flow profiles become significantly more complex. These alterations
in flow produce shear gradients, turbulence, flow separation, and
eddy formation, each of which can affect the atherogenic process.
Progression of the atherosclerotic lesion exacerbates flow distur-
bances, thus inducing a potential hazardous cycle of shear-
dependent acceleration of atherosclerosis.89
58
Shear effects on the endothelium
Endothelial cells in arteries are constantly exposed to dynamic
alterations in shear due to the pulsatile nature of blood flow.90
However, the rate of fluctuation in blood flow appears to be the
major parameter altering endothelial cell function. Endothelial cells
respond to flow changes through a variety of mechanotransduction
mechanisms, including mechanically gated ion channels and G pro-
teins, force-dependent alterations in membrane fluidity, nuclear
deformation, cytoskeletal-dependent junctional signaling, and via
primary cilia and the glycocalyx.91 These mechanosensory signaling
mechanisms are exquisitely sensitive to changes in wall shear stress,
leading to alterations in cell morphology, gene-expression profiles,
and increased adhesiveness.92
Atheromatous lesions in permissive flow environments for
leukocyte adhesion
The mechanisms by which disturbed blood flow influences leuko-
cyte adhesion mechanisms remain ill defined. In contrast to
platelets, leukocytes adhere preferentially at low shear regions in the
vasculature, typically at wall shear rates 1000/s. Whereas a
minimum threshold shear is required for leukocyte adhesion,93 there
is typically an inverse correlation between wall shear and the
efficiency of leukocyte recruitment, with optimal adhesion around
150/s.93 The site density of P- and E-selectin molecules also plays a
major role in regulating the efficiency of leukocyte adhesion;
therefore, the prevailing flow conditions and the degree of P-selectin
expression on the surface of activated endothelial cells and endothe-
lial-bound platelets are likely to be major determinants regulating
leukocyte recruitment to atherogenesis-prone sites.94 Experimental
models of disturbed blood flow lead to enhanced leukocyte accumu-
lation in low shear recirculation zones,95 supporting the concept that
flow disturbances may facilitate leukocyte recruitment to atheroscle-
rotic sites.
Impact of high shear and disturbed blood flow on platelet
adhesive function
The importance of shear forces in regulating platelet adhesive
function has long been recognized. At normal wall shear rates
(20-2000/s), shear does not stimulate the activation of platelets in
suspension. However at high stress rates ( 5000/s) as occurs in
stenosed atherosclerotic arteries, shear can directly induce platelet
activation.5 Based on in vitro findings, shear-induced platelet
activation is critically dependent upon the sequential binding of
VWF to GPIb and integrin IIb3, as well as on the release of
ADP.96 Shear-induced platelet activation is insensitive to COX
inhibitors, a finding that may partly explain the relative weak
antithrombotic effect of aspirin.97 In a complex shear environment
characterized by rapid phases of shear acceleration and deceleration,
platelet aggregation can occur independently of ADP and TxA2,98
raising the possibility that shear gradients can promote platelet
deposition and initial thrombus growth even in the presence of
aspirin and a P2Y12 inhibitor.
Conclusion
It has long been recognized that arterial thrombosis primarily
represents an exaggerated physiological hemostatic response at sites
of atherosclerotic plaque disruption. A similar concept can be
developed for the role of platelets in the initiation and propagation
of atherosclerosis. Platelets normally interact with endothelial cells
to preserve their physiological function and to enhance leukocyte
recruitment to sites of inflammation. An exaggeration of these
physiological responses at atherosclerosis-prone sites appears to
American Society of Hematology
play an important role in the pathogenesis of this disease. This
improved understanding of the role of platelets in the development
of atherothrombosis and elucidation of the key pathways involved
should stimulate further investigation into the possibility of target-
ing both the proinflammatory and prothrombotic function of plate-
lets to reduce the clinical complications of this major disease.
Acknowledgments
We thank Dr Simone Schoenwaelder for assistance with the
preparation of the figures. Z.S.K. is an National Health and Medical
Research Council (NHMRC) and National Heart Foundation post-
graduate scholarship holder. S.P.J. is a NHMRC Australia Fellow.
Disclosures
Conflict-of-interest disclosure: The authors declare no competing
financial interests. Off-label drug use: None disclosed.
Correspondence
Shaun P. Jackson, Australian Centre for Blood Diseases, 6th Level
Burnet Bldg, AMREP, 89 Commercial Rd, Melbourne, Victoria,
Australia 3004; Phone: 61-3-9903-0131; Fax: 61-3-9903-0228;
e-mail: shaun.jackson@monash.edu.
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