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 interactionsthroughout the entire vasculature irrespective of the prevailing hemodynamic conditions. This unique property ofplatelets is critical for their ability to arrest bleeding and promote vessel repair. Platelet adhesion under conditions ofhigh 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 orhemorrhagic complications. Whereas the central role of platelets in hemostasis and thrombosis has long beenrecognized and well defined, there is now a major body of evidence supporting an important proinflammatory functionfor platelets that is linked to host defense and a variety of autoimmune and inflammatory diseases. In the context of thevasculature, experimental evidence indicates that the proinflammatory function of platelets can regulate variousaspects of the atherosclerotic process, including its initiation and propagation. The mechanisms underlying theproatherogenic function of platelets are increasingly well defined and involve specific adhesive interactions betweenplatelets and endothelial cells at atherosclerotic-prone sites, leading to the enhanced recruitment and activation ofleukocytes. 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 responsethat accelerates atherosclerosis. These inflammatory processes typically occur in regions of the vasculatureexperiencing 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 theprothrombotic and proinflammatory function of platelets.

IntroductionPlatelets are anuclear cell fragments derived from the cytoplasm ofBM megakaryocytes1 that circulate in the bloodstream of humansfor 7-10 days. The primary hemostatic function of platelets has beenrecognized for more than a century, and the molecular processesregulating the adhesive function of platelets have been elucidated ingreat detail.2-4 In addition to preventing excess bleeding, plateletsalso play an important role in surveying and maintaining theintegrity of the endothelium, in part through the release of proangio-genic cytokines and growth factors. Dysregulated platelet-endothelial interactions have increasingly been recognized as animportant pathogenic mechanism in the development of atheroscle-rosis. In response to specific proinflammatory signals, endothelialcells become more adhesive toward platelets, stimulating theproduction of various platelet-derived inflammatory molecules thatprovide a positive feedback loop for further endothelial cellactivation. Endothelial-bound platelets are highly effective at recruit-ing leukocytes from flowing blood and also enhance leukocyteadhesion and transmigration to the site of the proinflammatorystimulus. Therefore, pathological derangement of these key interac-tions among platelets, endothelial cells, and leukocytes facilitatesthe inflammatory process that underlies the development ofatherosclerosis.

This review outlines the key pathways used by platelets to facilitatethe development of vascular inflammatory lesions linked to thedevelopment of atherosclerosis, with a focus on the mechanismsleading to dysregulation of the platelet-endothelium and platelet-

leukocyte interaction. The importance of local blood flow distur-bances in regulating the atherothrombotic process is briefly dis-cussed. A brief overview of the well-defined platelet adhesionmechanisms regulating platelet-vessel wall and platelet-plateletinteractions and a discussion of recent advances in the understand-ing of the platelet procoagulant response are presented first.

Hemostatic and prothrombotic function of plateletsUnder normal physiological conditions, platelets circulate in closeproximity to the endothelium without forming stable adhesioncontacts due to the anti-adhesive properties of quiescent endothelialcells. However, after vascular injury, platelets rapidly adhere to sitesof endothelial disruption to establish a hemostatic plug that preventsexcess blood loss. This process is critically dependent on theefficiency of platelet adhesion to the subendothelial matrix, as wellas on the ability of the platelets to undergo rapid biochemical andmorphological changes that support aggregation and the localizedactivation of the coagulation cascade.

Platelet recruitment (Figure 1)The mechanisms supporting platelet adhesion to the damaged vesselwall have been thoroughly investigated and well defined, so only abrief description will be provided here. After damage to the vesselwall, subendothelial matrix proteins that are highly reactive toplatelets, including collagen, VWF, fibronectin, and laminin, be-come exposed to the blood and immediately engage specificreceptors on the platelet surface. The contribution of specific ligandsand receptors mediating platelet adhesion is critically dependent on

ANTIPLATELET THERAPY

Hematology 2011 51

the prevailing blood-flow conditions. Under conditions of highshear stress, as encountered in arterioles and stenotic arteries, VWFplays a critical role in recruiting platelets from blood flow.5 Thistethering function of subendothelial VWF is dependent on theinteraction of its A1 domain with the glycoprotein Ib� (GPIb�)

component of the platelet GPIb-V-IX complex.6 Circulating plasmaVWF has limited binding potential for GPIb�, however, onceimmobilized onto subendothelial collagen (type I, III, and VI), theunfolded VWF macromolecule provides a linear array of A1domains that facilitate binding to multiple GPIb� receptors.7,8 The

Figure 1. Hemostatic and prothrombotic function of platelets under flow. (A) Platelet adhesion mechanisms operating under arterial flow. Afterdisruption of the endothelium, platelets are rapidly recruited from flowing blood via a tethering mechanism dependent on the interaction of plateletGPIb-V-IX and VWF. This adhesive bond has a rapid dissociation rate, resulting in platelet translocation on the vessel wall. (B) Platelet firm adhesionand aggregation. Translocating platelets engage collagen in the vessel wall through their adhesion receptors GPVI and �2�1. GPVI is the majorcollagen receptor inducing intracellular calcium flux necessary for stable platelet adhesion, cytoskeletal reorganization, �IIb�3 activation, and the releaseof 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. Locallygenerated thrombin also enhances platelet activation through proteolytic cleavage and activation of PARs. Activated platelets form stable aggregatesthrough �IIb�3 engagement of VWF and fibrinogen. These molecular pathways of amplification of platelet activation are the target of both clinicallyavailable and experimental therapeutic interventions in the treatment of atherothrombotic disease. (C) Stabilization of the platelet thrombus. The primaryhemostatic plug is consolidated by fibrin generation at the site of injury and throughout the developing thrombus. �-Thrombin generation at the injuredvessel wall is critically dependent on TF, whereas thrombin generation on the surface of activated platelets is likely to evolve both TF and contactfactor–dependent activation of blood coagulation. UHF indicates unfractionated heparin; LMWH, low-molecular-weight heparin; VKA, vitamin Kantagonist; PGH2, prostaglandin H2; AA, arachidonic acid, PLA2, phospholipase A2; IP3R, inositol trisphosphate receptor; ITAM, immunoreceptortyrosine-based activation motif; FcR�, Fc receptor gamma.

52 American Society of Hematology

bond between GPIb� and VWF has a rapid dissociation rate that isunable to support stable adhesion, requiring the contribution ofother ligand-receptor interactions to promote stable adhesion.9 Thefundamental importance of both VWF and GPIb� for the adhesivefunction of platelets is underscored by the severe bleeding disorderexperienced by patients with qualitative or quantitative defects inthese molecules.10

Firm adhesion and activation (Figure 1)Once tethered to the site of vascular injury, platelets form stableadhesion contacts with collagen and other matrix macromolecules.Platelet binding to collagen is mediated by GPVI11,12 and integrin�2�1,13 whereas fibronectin and laminin engage integrin �5�1 and�6�1, respectively.4 Isolated deficiency of any of these receptorsdoes not cause a severe bleeding disorder, suggesting considerableredundancy in the mechanisms regulating platelet-vessel wallinteractions. Once adherent, platelets undergo a remarkably com-plex series of morphological and biochemical changes that lead tothe release of platelet granule contents and up-regulation of theadhesive function of integrin �IIb�3. Activated �IIb�3 binds multipleligands, 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�IIb�3 leads to a major defect in the hemostatic function of plateletsand a severe bleeding disorder (Glanzmann thrombasthenia).19

Integrin �IIb�3 deficiency also provides protection against arterialthrombosis, thus pharmacological �IIb�3 antagonism represents ahighly effective antithrombotic approach.20,21

Soluble agonists amplifying platelet activation (Figure 1)Central to platelet activation is the generation and release of solubleagonists 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 fromplatelet dense granules. These endogenous agonists can act in anautocrine or paracrine manner to enhance platelet activation byengaging specific G protein coupled receptors; P2Y1

22 and P2Y1223

(ADP) and the thromboxane receptors TP� and TP�.24 ADP andTxA2 act in a cooperative manner to enhance platelet activation andthrombus formation, thus pharmacological targeting of TxA2

25

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, plateletsprovide a catalytic surface for the assembly of coagulation com-plexes necessary for �-thrombin generation. Thrombin is among themost potent stimulators of platelets through proteolytic cleavageand activation of platelet protease-activated receptors (PARs),specifically PAR1 and PAR4, on human platelets.29 Also central tothe hemostatic function of thrombin is its ability to generate fibrinpolymers, a key step stabilizing formed thrombi. Therefore, pharma-cological inhibitors of thrombin (eg, direct thrombin inhibitors),upstream coagulation proteases (vitamin K antagonists and factorXa [FXa] inhibitors), and, potentially, thrombin receptors (PAR1antagonists), represent effective strategies to limit arterial thrombusgrowth.27

Procoagulant function of platelets: emergingconceptsCurrent therapeutic strategies aimed at reducing thrombin’s effectsin vivo primarily target various components of the coagulation

cascade, thus affecting thrombin generation at the vessel wall aswell as within the body of thrombus. However, recent progress inunderstanding the mechanisms by which platelets support bloodcoagulation have raised the possibility that selectively inhibitingplatelet procoagulant function may provide a targeted approach tospecifically reduce thrombin generation within the thrombus.

Platelet regulation of the contact phase of bloodcoagulation (Figure 2)The generation of thrombin at the injured vessel wall is contingentupon the expression of tissue factor (TF) on the surface offibroblasts, smooth muscle cells, and potentially endothelial cellsand leukocytes. TF expression and activation helps to stabilize theactive conformation of FVIIa,30 triggering the extrinsic pathway ofblood coagulation. The mechanisms underlying the generation ofthrombin within the body of a developing thrombus remainscontroversial. Whereas TF is expressed on the surface of activatedleukocytes and microparticles and has been demonstrated to beincorporated within the thrombus, recent experimental evidence hassuggested a potentially important role for the contact phase of bloodcoagulation in promoting arterial thrombus growth. This rekindledinterest in the role of contact factors (eg, FXII, FXI, and high-molecular-weight kininogen) in promoting blood coagulation invivo stemmed from the observation that FXII-deficient mice areprotected 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 inarterial thrombosis. Given that humans with complete FXII defi-ciency do not have a significant hemostatic defect, this has raised thepossibility that therapeutic targeting of this process may lead to thedevelopment of safer antithrombotics. How platelets activate thecontact phase of coagulation during thrombus development hasremained a vexing issue.35 Recently, Muller et al identified inor-ganic polyphosphate released from activated platelets as a majorregulator of FXII activation.36 Pharmacologic exploitation of thispathway may enable targeting of platelet polyphosphate by phospha-tases to reduce thrombotic risk while leaving hemostatic responsesintact.

Identification of the elusive phospholipid scramblaseThe contribution of platelet-derived phospholipids to thrombingeneration has long been recognized; however, the biologicalmechanisms regulating this procoagulant platelet function haveremained poorly defined. In resting platelets, membrane phospholip-ids are asymmetrically distributed: phosphatidylcholine and sphin-gomyelin are primarily localized in the outer leaflet of the plasmamembrane, whereas phosphatidylethanolamine and phosphatidylser-ine (PS) are largely confined to the inner leaflet. This asymmetry ismaintained through the action of two membrane lipid transporters,flippase and floppase.37,38 After platelet stimulation by potentagonists, this asymmetry is lost due to activation of Ca2�-dependentphospholipid scramblases that bidirectionally transport phospholip-ids.39 Scramblase activity therefore results in the expression ofnegatively charged phospholipids, especially PS, on the outer leafletof the platelet membrane, thus providing the requisite surface forassembly of coagulation factors required for thrombin generation(Figure 2). Defects in platelet PS expression are rare; however, inaffected individuals, this leads to a major hemostatic disturbance, asdescribed in Scott syndrome.40 Until recently, the identity of thephospholipid scramblase had remained elusive, but recent studieshave identified a plasma membrane protein transmembrane protein16F (TMEM16F) that confers Ca2�-dependent scrambling of phos-pholipids. A mutation in the TMEM16F gene resulting in premature

Hematology 2011 53

termination of the protein has been demonstrated in a patient withScott syndrome.41

Cell death and the procoagulant function of platelets(Figure 2)Procoagulant platelets expressing PS exhibit distinct morphologicand biochemical features relative to fully activated platelets. Manyof these changes are indicative of dead or dying cells, and includemembrane blebbing, microvesiculation, activation of intracellularproteases, and the cleavage of cytoskeletal proteins, PS surfaceexposure, and alterations in mitochondrial membrane potential.42

These results suggest that the pathways regulating cell death inplatelets may play an important role in regulating the plateletprocoagulant phenotype. Programmed cell death is orchestrated byseveral pathways, including apoptosis, necrosis, and autophagy.Apoptosis is an energy-dependent process contingent upon caspaseactivation and is critical for the removal of senescent cells. Necrosisis 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 plateletstimulation by potent agonists initiates a tightly regulated cell deathpathway mediated by sustained high levels of cytosolic Ca2�,culminating in distinct morphological and biochemical changes thatare consistent with a necrotic cell death pathway.43 Direct inductionof apoptosis can also induce PS expression and platelet procoagu-lant function,43 raising the possibility that multiple cell deathpathways may regulate thrombin generation on the surface ofplatelets. In addition to promoting thrombin generation, procoagu-lant platelets may also contribute to the inflammatory response bygenerating high levels of the proinflammatory lipid platelet-activating factor (PAF) and by enhancing leukocyte adhesion.44

Proinflammatory role of plateletsPromiscuous plateletsThe growing list of pathophysiological processes in which plateletshave a proposed role is in part a reflection of the large number of

Figure 2. Procoagulant function of platelets. (A) Plasma membrane phospholipids are asymmetrically distributed in resting platelets, with PSconfined 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 therequisite 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, andsubstrate proteolysis. Necrotic cell death can be initiated by elevated cytosolic Ca2�, causing a loss in mitochondrial membrane potential andsubsequent formation of a mitochondrial permeability transition pore (mPTP). mPTP formation results in bioenergetic failure through ATP depletion withconsequent reactive oxygen species (ROS) generation, leading to loss of membrane integrity. (C) Platelets can also contribute to thrombin generationvia activation of FXII through the release of polyphosphate from dense granules. (D) Whether there are additional cell death pathways regulating PSexposure and the procoagulant function of platelets remains unclear. PC indicates phosphatidylcholine.

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different cell types with which platelets can interact. These includeendothelial cells, neutrophils, monocytes, dendritic cells, cytotoxicT-lymphocytes, malaria-infected red blood cells, and various tumorcells. In the context of atherosclerosis, the interaction of plateletswith endothelial cells and leukocytes appears to be important for theinitiation and propagation of inflammatory processes in the arterialwall.45

Platelet-endothelial cell adhesive interactions (Figure 3)While platelets continuously circulate in close proximity to theendothelium, under physiological conditions, they typically do notform sustained adhesive interactions. The anti-adhesive phenotypeof endothelial cells is controlled by the three intrinsic pathways; thenitric 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 stimulifacilitate sustained endothelial-platelet adhesion by perturbing theseanti-adhesive mechanisms and increasing surface expression ofendothelial adhesion molecules.47 The adhesion of platelets to“inflamed endothelium” involves a similar coordinated multistepprocess as occurs in hemostasis and thrombosis, including platelettethering, surface translocation, and firm adhesion.48 Initiation of theinflammatory insult to the endothelium in atherosclerosis is partlydue to the accumulation of modified oxidized lipoprotein particlesin the setting of hyperlipidemia.49 This leads to surface expressionof endothelial selectins, including P- and E-selectin, as well asmembrane expression of VWF.48,50 The platelet counterreceptors

for P-selectin include GPIb� and P-selectin glycoprotein-1 (PSGL-1),51,52 which support tethering and rolling, but not firm adhesion oninflamed endothelium. Whereas there is some uncertainty as to theexact mechanism supporting stable platelet adhesion to activatedendothelium, in vitro and in vivo models suggest that binding of�IIb�3 to fibrinogen bound to endothelial �v�3 or ICAM-1 facilitatesfirm platelet adhesion and activation.53,54

Chemical signalsPlatelets 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-derivedbioactive substances alter the chemotactic and adhesive propertiesof the endothelial cells, enabling monocytes and other leukocytes toadhere and transmigrate through the endothelium to sites ofinflammation.48,56 In vivo models of atherogenesis suggest thatthese interactions between endothelial cells and activated plateletsare likely to be critical in the initiation of atherosclerosis.57,58

IL-I� (Figure 3). IL-I� is a platelet derived cytokine that has beenpostulated to be a principal mediator of platelet-induced endothelialactivation.59,60 It is not stored in platelet granules, but is believed tobe synthesized by platelets from constitutional prepackaged mRNAseveral hours after thrombin stimulation or integrin-mediated adhe-sion.61 IL-I� stimulation of endothelial cells induces secretion ofIL-6 and IL-8 and significantly increases the endothelial surfaceexpression of ICAM-1, �v�3, and monocyte chemotactic protein-1

Figure 3. Platelet-endothelial cell interactions. (A) The healthy endothelium. The anti-adhesive phenotype of endothelial cells is maintained through3 intrinsic pathways: the ecto-ADPase/CD39/NTPDase pathway, which metabolizes ADP, and the PGI2 and NO pathways, which inhibit plateletactivation through the stimulation of cAMP and cGMP production, respectively. (B) Endothelial activation in hyperlipemia. In a hyperlipidemic milieu, theendothelium becomes inflamed as a result of modified lipoprotein particles and reactive oxygen species (ROS) accumulating in the intima. This leads tothe expression of adhesive ligands (VWF and P- and E-selectins) on the endothelium. (C) Platelet adhesion to the inflamed endothelium and plateletendothelial cross-talk. The expression of VWF and P-selectin on the endothelial surface supports platelet tethering and rolling. Subsequently, stableplatelet adhesion occurs through fibrinogen-�IIb�3 complexes binding to �v�3 or ICAM-1 on endothelial cells. Adherent platelets secrete numerousbioactive substances that alter the chemotactic and adhesive properties of the endothelial cells. Platelet-derived IL-1� induces endothelial secretion ofIL-6 and IL-8 and surface expression of ICAM-1, �v�3, and MCP-1. Platelet CD40 ligand binds CD40 on endothelial cells, resulting in up-regulation ofadhesive molecules (ICAM-1, VCAM-1, and E- and P-selectin), cytokine and TF release, and reduction in NO synthesis. Ecto ADPase indicatesecto-adenosine diphosphatase; NTPDase, nucleoside triphosphate diphosphohydrolase.

Hematology 2011 55

(MCP-1).48,60,62 ICAM-1 and MCP-1 up-regulation by endothelialcells in response to IL-I� is dependent on the activation of NF-�B.63

Through this potential mechanism, platelets are able to significantlyenhance monocyte and neutrophil adhesion to the endothelium.

CD40 ligand (CD40L-CD154; Figure 3). CD40 ligand is animportant platelet-derived bioactive mediator of endothelial cellactivation. CD40L is a transmembrane protein with structuralhomology to TNF-�.64 CD40L was initially identified on the surfaceof activated CD4� T cells and is central to the function of thehumoral immune system by supporting B-cell differentiation andimmunoglobulin class switching.65 It was subsequently demon-strated that platelets are a rich source of CD40L,66 with � 95% ofcirculating CD40L being of platelet origin.67 After release fromplatelets, CD40L interacts which CD40 expressed on numerous celltypes, 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 andTF.69 CD40L inhibits endothelial NO production by destabilizingNO synthase mRNA64,70; therefore, CD40L binding to endothelialcells results in a proatherogenic and proinflammatory phenotype.64

Platelet-leukocyte cross-talk in atherogenesisThe 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 theinflamed endothelium may enhance leukocyte recruitment, activa-tion, and transmigration, thereby enhancing the inflammatoryprocesses underlying atherosclerosis.

Leukocyte recruitment to endothelial-bound platelets is a coordi-nated, multistep process involving selectin-mediated tethering androlling, followed by �2 integrin–mediated stable adhesion (Figure

Table 1. Bioactive platelet mediators underlying atherothrombosis

Localization within theplatelet Released molecule Target cell Function

Dense granules ADP Platelet P2Y1 and P2Y12 Platelet activationATP Platelet P2X1 Platelet activation and sensitization to other

agonists5HT Platelet 5HT2A Weak platelet agonist

Endothelium 5HT2B NO synthesis: vasodilation, platelet inhibitorVascular smooth muscle VasoconstrictionMonocytes T-cell activation: proinflammatory

�� granulesAdhesion receptors �IIb�3 Platelet Platelet aggregation, platelet-endothelial, and

platelet-leukocyte adhesionCoagulation factors FV ProcoagulantAdhesive ligands VWF Platelet Platelet-endothelium and platelet-vessel wall

tethering and platelet aggregationFibrinogen Platelet Platelet aggregation, fibrin clot formation, and

platelet-leukocyte adhesionFibronectin Platelet Platelet-endothelial and platelet-leukocyte

adhesionP-selectin Platelets, endothelial cell and

leukocytes–PSGL-1Adhesion of leukocytes to platelets and

endothelial cellsChemokines CXCL4 (PF-4) Leukocytes Synergizes with other cytokines to promote

leukocyte adhesion and monocytedifferentiation

CXCL7 (PBP, �-TG, CTAP-III,and NAP-2)

Leukocytes Enhances neutrophil and monocyte adhesionand neutrophil transendothelial migration

CCL5 (RANTES) Endothelium/leukocytes Promotes monocytes adhesion to theendothelium and chemokine synthesis

Growth factors PDGF Vascular smooth musclecells

Stimulates vascular smooth muscle cellmigration and proliferation associated withintimal hyperplasia

Synthesized cytokines IL-1� Endothelial cells Up-regulation of leukocyte adhesion moleculesand stimulation of endothelial release ofproinflammatory cytokines

�� granule and plateletmembrane

CD40 Ligand Endothelial cell CD40 Upregulation of leukocyte adhesion moleculesand stimulation of endothelial release ofproinflammatory cytokines

Proinflammatory lipids TxA2 Platelets Platelet activationLeukotriene B4 Leukocytes (BLT-1 and

BLT-2)Chemo-attractant and promotion of monocyte

differentiation into foam cells and neutrophilactivation

PAF Platelet/leukocytes Platelet agonist and promotes neutrophiladhesion

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4). Platelet P-selectin has a central role in mediating the initialcapture and rolling of leukocytes through interactions with its keycounterreceptor P-selectin glycoprotein 1 (PSGL-1).51,72 Ligation ofPSGL-1 induces signaling events culminating in the activation ofthe leukocyte �2 integrins, including macrophage antigen-1 (Mac-1,�M�2) and lymphocyte function-associated antigen-1 (LFA-1, �L�2),which are required to mediate stable leukocyte adhesion. Mac-1 isthe principal �2 integrin that facilitates firm adhesion onto thesurface of platelets via its binding to multiple ligands, includingGPIb�,73 ICAM-2,74 and junctional adhesion molecule-3 (JAM-3),75 as well as through fibrinogen bound to �IIb�3.48,76

Platelet activation with consequent degranulation results in theliberation 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 numerousbiological 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 hasseveral molecular variants, including the inactive precursor, connec-tive tissue-activating peptide III (CTAP-III), which is enzymaticallyconverted 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 induceneutrophil and monocyte adhesion in vitro and neutrophil transendo-thelial migration.78,80 The chemotactic properties of PF4 on its ownare controversial because it does not exert classical chemokine

Figure 4. Platelet-leukocyte interactions promoting atherogenesis. (A) Platelet-mediated leukocyte recruitment and adhesion. Leukocyterecruitment to endothelial-bound platelets occurs in a multistep coordinated process. Initial tethering of leukocytes is mediated by the interaction ofP-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 �IIb�3. 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 enhanceleukocyte 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 membraneassociated serine-protease cathepsin G. NAP-2 enhances leukocyte adhesion and neutrophil transendothelial migration. Up-regulation of COX-2 inmonocytes via pathways involving IL-I� and P-selectin results in increased production of proinflammatory mediators, including PAF, leukotrienes, andTxA2 via transcellular eicosanoid metabolism. P-sel indicates P-selectin; AA, arachidonic acid.

Hematology 2011 57

functions at submicromolar concentrations.78,81 However, PF4 act-ing in concert with other chemokines such as the platelet-derivedCC chemokine regulated upon activation normal T-cell expressedand secreted (RANTES) may enhance leukocyte adhesion andpromote monocyte differentiation and atherosclerosis. These PF4-RANTES heterodimers have been proposed to represent potentialtherapeutic targets in the treatment of atherosclerosis.82

CC chemokine RANTES (Figure 4)The proinflammatory CC chemokine RANTES is released fromactivated platelets and deposited on activated endothelium, where itis able to promote adhesion of monocytes.83 A prerequisite for the invivo activity of RANTES function is for it to bind to glycosamino-glycans and to form higher-order oligomers.84 Further, through itsinteractions with other modulators, including MCP-4, PF4, andP-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 forthe 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 adifferent cytokine profile, including MCP-1 and IL-8.85 Plateletactivation results in the release of microparticles that are able tofacilitate 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. Theadhesion of platelets to leukocytes and the endothelium results intranscellular metabolism of eicosanoids, leading to the local synthe-sis of proinflammatory lipids such as PAF, leukotrienes, andTxA2.87 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 andIL-I�. This up-regulation of COX-2 enables further synthesis ofproinflammatory eicosanoids.88 PAF is a potent platelet agonist andinflammatory mediator and has also been shown to regulate firmneutrophil adhesion on the surface of immobilized spread platelets.44

Atherothrombosis and disturbed blood flowAn important determinant influencing platelet-endothelial and plate-let-leukocyte interactions at atherosclerotic-prone sites is the localblood flow conditions. It is well established that atheroscleroticlesions develop at arterial branch points, which are typically areas oflow shear and disturbed flow. Altered flow has a significant effect onthe adhesive properties of platelets and leukocytes, as well on themorphology and function of endothelial cells. Rheological factorsalso contribute to the deposition of lipids and other modulators ofatherosclerosis.

Throughout much of the vasculature, blood flow is laminar, withadjacent fluid layers traveling parallel to each other but at differentvelocities because of the fluid drag exerted by the vessel wall. Thisphenomenon leads to the generation of shear forces betweenadjacent fluid planes. These flow profiles apply to straight, healthyvessels—at arterial branch points, curvatures, and areas of stenosis,flow profiles become significantly more complex. These alterationsin flow produce shear gradients, turbulence, flow separation, andeddy 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

Shear effects on the endotheliumEndothelial cells in arteries are constantly exposed to dynamicalterations in shear due to the pulsatile nature of blood flow.90

However, the rate of fluctuation in blood flow appears to be themajor parameter altering endothelial cell function. Endothelial cellsrespond to flow changes through a variety of mechanotransductionmechanisms, including mechanically gated ion channels and G pro-teins, force-dependent alterations in membrane fluidity, nucleardeformation, cytoskeletal-dependent junctional signaling, and viaprimary cilia and the glycocalyx.91 These mechanosensory signalingmechanisms 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 forleukocyte adhesionThe mechanisms by which disturbed blood flow influences leuko-cyte adhesion mechanisms remain ill defined. In contrast toplatelets, leukocytes adhere preferentially at low shear regions in thevasculature, typically at wall shear rates � 1000/s. Whereas aminimum threshold shear is required for leukocyte adhesion,93 thereis typically an inverse correlation between wall shear and theefficiency of leukocyte recruitment, with optimal adhesion around150/s.93 The site density of P- and E-selectin molecules also plays amajor role in regulating the efficiency of leukocyte adhesion;therefore, the prevailing flow conditions and the degree of P-selectinexpression on the surface of activated endothelial cells and endothe-lial-bound platelets are likely to be major determinants regulatingleukocyte recruitment to atherogenesis-prone sites.94 Experimentalmodels of disturbed blood flow lead to enhanced leukocyte accumu-lation in low shear recirculation zones,95 supporting the concept thatflow disturbances may facilitate leukocyte recruitment to atheroscle-rotic sites.

Impact of high shear and disturbed blood flow on plateletadhesive functionThe importance of shear forces in regulating platelet adhesivefunction has long been recognized. At normal wall shear rates(20-2000/s), shear does not stimulate the activation of platelets insuspension. However at high stress rates (� 5000/s) as occurs instenosed atherosclerotic arteries, shear can directly induce plateletactivation.5 Based on in vitro findings, shear-induced plateletactivation is critically dependent upon the sequential binding ofVWF to GPIb� and integrin �IIb�3, as well as on the release ofADP.96 Shear-induced platelet activation is insensitive to COXinhibitors, a finding that may partly explain the relative weakantithrombotic effect of aspirin.97 In a complex shear environmentcharacterized 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 plateletdeposition and initial thrombus growth even in the presence ofaspirin and a P2Y12 inhibitor.

ConclusionIt has long been recognized that arterial thrombosis primarilyrepresents an exaggerated physiological hemostatic response at sitesof atherosclerotic plaque disruption. A similar concept can bedeveloped for the role of platelets in the initiation and propagationof atherosclerosis. Platelets normally interact with endothelial cellsto preserve their physiological function and to enhance leukocyterecruitment to sites of inflammation. An exaggeration of thesephysiological responses at atherosclerosis-prone sites appears to

58 American Society of Hematology

play an important role in the pathogenesis of this disease. Thisimproved understanding of the role of platelets in the developmentof atherothrombosis and elucidation of the key pathways involvedshould 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.

AcknowledgmentsWe thank Dr Simone Schoenwaelder for assistance with thepreparation of the figures. Z.S.K. is an National Health and MedicalResearch Council (NHMRC) and National Heart Foundation post-graduate scholarship holder. S.P.J. is a NHMRC Australia Fellow.

DisclosuresConflict-of-interest disclosure: The authors declare no competingfinancial interests. Off-label drug use: None disclosed.

CorrespondenceShaun P. Jackson, Australian Centre for Blood Diseases, 6th LevelBurnet 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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