ANTIPLATELET THERAPY

The Role of Platelets in Atherothrombosis

Zane S. Kaplan1 and Shaun P. Jackson1
Centre for Blood Diseases, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Australia
1Australian

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 uidity and to prevent thrombotic or hemorrhagic complications. Whereas the central role of platelets in hemostasis and thrombosis has long been recognized and well dened, there is now a major body of evidence supporting an important proinammatory function for platelets that is linked to host defense and a variety of autoimmune and inammatory diseases. In the context of the vasculature, experimental evidence indicates that the proinammatory 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 dened and involve specic 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, proinammatory molecules, and other biological response modulators, the interaction among platelets, endothelial cells, and leukocytes establishes a localized inammatory response that accelerates atherosclerosis. These inammatory processes typically occur in regions of the vasculature experiencing low shear and perturbed blood ow, a permissive environment for leukocyte-platelet and leukocyteendothelial interactions. Therefore, the concept has emerged that platelets are a central element of the atherothrombotic process and that future therapeutic strategies to combat this disease need to take into consideration both the prothrombotic and proinammatory function of platelets.

Introduction
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 proangiogenic cytokines and growth factors. Dysregulated plateletendothelial interactions have increasingly been recognized as an important pathogenic mechanism in the development of atherosclerosis. In response to specic proinammatory signals, endothelial cells become more adhesive toward platelets, stimulating the production of various platelet-derived inammatory molecules that provide a positive feedback loop for further endothelial cell activation. Endothelial-bound platelets are highly effective at recruiting leukocytes from owing blood and also enhance leukocyte adhesion and transmigration to the site of the proinammatory stimulus. Therefore, pathological derangement of these key interactions among platelets, endothelial cells, and leukocytes facilitates the inammatory process that underlies the development of atherosclerosis. This review outlines the key pathways used by platelets to facilitate the development of vascular inammatory lesions linked to the development of atherosclerosis, with a focus on the mechanisms leading to dysregulation of the platelet-endothelium and platelet-

leukocyte interaction. The importance of local blood ow disturbances in regulating the atherothrombotic process is briey discussed. A brief overview of the well-dened platelet adhesion mechanisms regulating platelet-vessel wall and platelet-platelet interactions and a discussion of recent advances in the understanding of the platelet procoagulant response are presented rst.

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 efciency 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 dened, 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, bronectin, and laminin, become exposed to the blood and immediately engage specic receptors on the platelet surface. The contribution of specic ligands and receptors mediating platelet adhesion is critically dependent on

Hematology 2011

51

Figure 1. Hemostatic and prothrombotic function of platelets under ow. (A) Platelet adhesion mechanisms operating under arterial ow. After disruption of the endothelium, platelets are rapidly recruited from owing 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 rm adhesion and aggregation. Translocating platelets engage collagen in the vessel wall through their adhesion receptors GPVI and 2 1. GPVI is the major collagen receptor inducing intracellular calcium ux necessary for stable platelet adhesion, cytoskeletal reorganization, IIb 3 activation, and the release of the soluble agonists (ADP and TxA2). These agonists act through specic 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 IIb 3 engagement of VWF and brinogen. These molecular pathways of amplication 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 brin 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-ow conditions. Under conditions of high shear stress, as encountered in arterioles and stenotic arteries, VWF plays a critical role in recruiting platelets from blood ow.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

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 specically reduce thrombin generation within the thrombus.

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 13 whereas bronectin and laminin engage integrin 2 1, 5 1 and 4 6 1, respectively. Isolated deciency 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 complex series of morphological and biochemical changes that lead to the release of platelet granule contents and up-regulation of the adhesive function of integrin IIb 3. Activated IIb 3 binds multiple ligands, including VWF,14,15 brinogen,16 brin and bronectin,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 platelets and a severe bleeding disorder (Glanzmann thrombasthenia).19 Integrin IIb 3 deciency also provides protection against arterial thrombosis, thus pharmacological IIb 3 antagonism represents a highly effective antithrombotic approach.20,21

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 broblasts, smooth muscle cells, and potentially endothelial cells and leukocytes. TF expression and activation helps to stabilize the 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 suggested a potentially important role for the contact phase of blood coagulation in promoting arterial thrombus growth. This rekindled interest in the role of contact factors (eg, FXII, FXI, and highmolecular-weight kininogen) in promoting blood coagulation in vivo stemmed from the observation that FXII-decient mice are protected from vasoocclusive thrombi in arterial thrombosis models.31 Similarly, FXI deciency32,33 and pharmacological antagonism34 of the contact phase of coagulation leads to a similar defect in arterial thrombosis. Given that humans with complete FXII deciency do not have a signicant 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 identied inorganic 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 phosphatases to reduce thrombotic risk while leaving hemostatic responses intact.

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 cyclooxygenase (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 specic 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

Identication 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 dened. In resting platelets, membrane phospholipids are asymmetrically distributed: phosphatidylcholine and sphingomyelin are primarily localized in the outer leaet of the plasma membrane, whereas phosphatidylethanolamine and phosphatidylserine (PS) are largely conned to the inner leaet. This asymmetry is maintained through the action of two membrane lipid transporters, ippase and oppase.37,38 After platelet stimulation by potent agonists, this asymmetry is lost due to activation of Ca2 -dependent phospholipid scramblases that bidirectionally transport phospholipids.39 Scramblase activity therefore results in the expression of negatively charged phospholipids, especially PS, on the outer leaet 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 identied a plasma membrane protein transmembrane protein 16F (TMEM16F) that confers Ca2 -dependent scrambling of phospholipids. A mutation in the TMEM16F gene resulting in premature

Thrombin (Figure 1)
In addition to the synthesis and release of soluble agonists, platelets provide a catalytic surface for the assembly of coagulation complexes necessary for -thrombin generation. Thrombin is among the most potent stimulators of platelets through proteolytic cleavage and activation of platelet protease-activated receptors (PARs), specically PAR1 and PAR4, on human platelets.29 Also central to the hemostatic function of thrombin is its ability to generate brin polymers, a key step stabilizing formed thrombi. Therefore, pharmacological 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

53

Figure 2. Procoagulant function of platelets. (A) Plasma membrane phospholipids are asymmetrically distributed in resting platelets, with PS conned to the inner leaet. 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 leaet 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 typied by bioenergetic failure of the cell resulting in membrane

instability and the release of intracellular contents into the extracellular 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 procoagulant 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, procoagulant platelets may also contribute to the inammatory response by generating high levels of the proinammatory lipid plateletactivating factor (PAF) and by enhancing leukocyte adhesion.44

Proinammatory role of platelets Promiscuous platelets

The growing list of pathophysiological processes in which platelets have a proposed role is in part a reection 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 inamed as a result of modied 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 inamed 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 brinogen- IIb 3 complexes binding to v 3 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, v 3, 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 inammatory processes in the arterial wall.45

for P-selectin include GPIb and P-selectin glycoprotein-1 (PSGL1),51,52 which support tethering and rolling, but not rm adhesion on inamed 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 IIb 3 to brinogen bound to endothelial v 3 or ICAM-1 facilitates rm platelet adhesion and activation.53,54

Platelet-endothelial cell adhesive interactions (Figure 3)

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 pathway, and the ecosanoid-arachidonic acid-prostacyclin (PGI2) pathway.46 It is increasingly recognized that inammatory 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 inamed endothelium involves a similar coordinated multistep process as occurs in hemostasis and thrombosis, including platelet tethering, surface translocation, and rm adhesion.48 Initiation of the inammatory insult to the endothelium in atherosclerosis is partly due to the accumulation of modied 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

Chemical signals
Platelets activated through their interactions with inamed endothelium release a vast array of inammatory 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 inammation.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 adhesion.61 IL-I stimulation of endothelial cells induces secretion of IL-6 and IL-8 and signicantly increases the endothelial surface expression of ICAM-1, v 3, and monocyte chemotactic protein-1

Hematology 2011

55

Table 1. Bioactive platelet mediators underlying atherothrombosis Localization within the platelet Dense granules ADP ATP 5HT Released molecule Target cell Platelet P2Y1 and P2Y12 Platelet P2X1 Platelet 5HT2A Endothelium 5HT2B Vascular smooth muscle Monocytes Platelet Function Platelet activation Platelet activation and sensitization to other agonists Weak platelet agonist NO synthesis: vasodilation, platelet inhibitor Vasoconstriction T-cell activation: proinammatory Platelet aggregation, platelet-endothelial, and platelet-leukocyte adhesion Procoagulant Platelet-endothelium and platelet-vessel wall tethering and platelet aggregation Platelet aggregation, brin clot formation, and platelet-leukocyte adhesion Platelet-endothelial and platelet-leukocyte adhesion Adhesion of leukocytes to platelets and endothelial cells Synergizes with other cytokines to promote leukocyte adhesion and monocyte differentiation Enhances neutrophil and monocyte adhesion and neutrophil transendothelial migration Promotes monocytes adhesion to the endothelium and chemokine synthesis Stimulates vascular smooth muscle cell migration and proliferation associated with intimal hyperplasia Up-regulation of leukocyte adhesion molecules and stimulation of endothelial release of proinammatory cytokines

granules Adhesion receptors Coagulation factors Adhesive ligands

IIb 3

FV VWF Fibrinogen Fibronectin P-selectin

Platelet Platelet Platelet Platelets, endothelial cell and leukocytesPSGL-1 Leukocytes

Chemokines

CXCL4 (PF-4)

CXCL7 (PBP, -TG, CTAP-III, and NAP-2) CCL5 (RANTES) Growth factors PDGF

Leukocytes Endothelium/leukocytes Vascular smooth muscle cells Endothelial cells

Synthesized cytokines

IL-1

granule and platelet membrane CD40 Ligand Endothelial cell CD40 Upregulation of leukocyte adhesion molecules and stimulation of endothelial release of proinammatory cytokines Platelet activation Chemo-attractant and promotion of monocyte differentiation into foam cells and neutrophil activation Platelet agonist and promotes neutrophil adhesion

Proinammatory lipids

TxA2 Leukotriene B4

Platelets Leukocytes (BLT-1 and BLT-2) Platelet/leukocytes

PAF

(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 signicantly enhance monocyte and neutrophil adhesion to the endothelium.

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 proinammatory phenotype.64

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 identied 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 demonstrated 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, broblasts, and platelets.64,68 In endothelial cells, CD40L binding

Platelet-leukocyte cross-talk in atherogenesis

The central role of leukocytes, especially monocytes and T lymphocytes, in the development of atherosclerosis is well established.71 There is increasing evidence that activated platelets adherent to the inamed endothelium may enhance leukocyte recruitment, activation, and transmigration, thereby enhancing the inammatory processes underlying atherosclerosis. Leukocyte recruitment to endothelial-bound platelets is a coordinated, 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 brinogen 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 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 proinammatory 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, M 2) and lymphocyte function-associated antigen-1 (LFA-1, L 2), which are required to mediate stable leukocyte adhesion. Mac-1 is the principal 2 integrin that facilitates rm adhesion onto the surface of platelets via its binding to multiple ligands, including GPIb ,73 ICAM-2,74 and junctional adhesion molecule-3 (JAM3),75 as well as through brinogen bound to IIb 3.48,76 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, connective 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 serineprotease cathepsin G.79 NAP-2 has been demonstrated to induce neutrophil and monocyte adhesion in vitro and neutrophil transendothelial 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 acting 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 PF4RANTES heterodimers have been proposed to represent potential therapeutic targets in the treatment of atherosclerosis.82

Shear effects on the endothelium

Endothelial cells in arteries are constantly exposed to dynamic alterations in shear due to the pulsatile nature of blood ow.90 However, the rate of uctuation in blood ow appears to be the major parameter altering endothelial cell function. Endothelial cells respond to ow changes through a variety of mechanotransduction mechanisms, including mechanically gated ion channels and G proteins, force-dependent alterations in membrane uidity, 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 proles, and increased adhesiveness.92

CC chemokine RANTES (Figure 4)

The proinammatory 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 glycosaminoglycans 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 inammation, atherogenesis, and vascular remodeling after injury. Interestingly, Pselectin engagement with PSGL-1 on monocytes is insufcient for the induction of MCP-1, requiring the cooperation of plateletderived RANTES for chemokine synthesis by monocytes.85 Further, monocytes costimulated by RANTES and P-selectin secrete a different cytokine prole, 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 inamed and atherosclerotic endothelium, thus promoting monocyte adhesion.86

Atheromatous lesions in permissive ow environments for leukocyte adhesion

The mechanisms by which disturbed blood ow inuences leukocyte adhesion mechanisms remain ill dened. 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 efciency 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 efciency of leukocyte adhesion; therefore, the prevailing ow conditions and the degree of P-selectin expression on the surface of activated endothelial cells and endothelial-bound platelets are likely to be major determinants regulating leukocyte recruitment to atherogenesis-prone sites.94 Experimental models of disturbed blood ow lead to enhanced leukocyte accumulation in low shear recirculation zones,95 supporting the concept that ow disturbances may facilitate leukocyte recruitment to atherosclerotic sites.

Proinammatory lipids (Figure 4)

In addition to being storage houses for cytokines and chemokines, platelets also generate lipid-derived inammatory mediators. The adhesion of platelets to leukocytes and the endothelium results in transcellular metabolism of eicosanoids, leading to the local synthesis of proinammatory lipids such as PAF, leukotrienes, and TxA2.87 The imbalance of inammatory mediators is further exacerbated by monocyte up-regulation of COX-2 expression via interactions involving P-selectin derived from activated platelets and IL-I . This up-regulation of COX-2 enables further synthesis of proinammatory eicosanoids.88 PAF is a potent platelet agonist and inammatory mediator and has also been shown to regulate rm neutrophil adhesion on the surface of immobilized spread platelets.44

Impact of high shear and disturbed blood ow 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 ndings, shear-induced platelet activation is critically dependent upon the sequential binding of VWF to GPIb and integrin IIb 3, as well as on the release of ADP.96 Shear-induced platelet activation is insensitive to COX inhibitors, a nding 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.

Atherothrombosis and disturbed blood ow

An important determinant inuencing platelet-endothelial and platelet-leukocyte interactions at atherosclerotic-prone sites is the local blood ow conditions. It is well established that atherosclerotic lesions develop at arterial branch points, which are typically areas of low shear and disturbed ow. Altered ow has a signicant 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 ow is laminar, with adjacent uid layers traveling parallel to each other but at different velocities because of the uid drag exerted by the vessel wall. This phenomenon leads to the generation of shear forces between adjacent uid planes. These ow proles apply to straight, healthy vesselsat arterial branch points, curvatures, and areas of stenosis, ow proles become signicantly more complex. These alterations in ow produce shear gradients, turbulence, ow separation, and eddy formation, each of which can affect the atherogenic process. Progression of the atherosclerotic lesion exacerbates ow disturbances, thus inducing a potential hazardous cycle of sheardependent acceleration of atherosclerosis.89

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 inammation. An exaggeration of these physiological responses at atherosclerosis-prone sites appears to

58

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 targeting both the proinammatory and prothrombotic function of platelets to reduce the clinical complications of this major disease.

15.

16.

Acknowledgments
We thank Dr Simone Schoenwaelder for assistance with the preparation of the gures. Z.S.K. is an National Health and Medical Research Council (NHMRC) and National Heart Foundation postgraduate scholarship holder. S.P.J. is a NHMRC Australia Fellow. 17.

18.

Disclosures
Conict-of-interest disclosure: The authors declare no competing nancial interests. Off-label drug use: None disclosed.

19.

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.

20.

21.

References
1. Junt T, Schulze H, Chen Z, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science. 2007;317(5845): 1767-1770. 2. Andrews RK, Lopez JA, Berndt MC. Molecular mechanisms of platelet adhesion and activation. Int J Biochem Cell Biol. 1997;29(1):91-105. 3. Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circ Res. 2007;100(12):1673-1685. 4. Ruggeri ZM. Platelet adhesion under ow. Microcirculation. 2009;16(1):58-83. 5. Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto brinogen or translocation on von Willebrand factor. Cell. 1996;84(2):289-297. 6. Ruggeri ZM. Structure and function of von Willebrand factor. Thromb Haemost. 1999;82(2):576-584. 7. Siedlecki CA, Lestini BJ, Kottke-Marchant KK, Eppell SJ, Wilson DL, Marchant RE. Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996;88(8):2939-2950. 8. Barg A, Ossig R, Goerge T, et al. Soluble plasma-derived von Willebrand factor assembles to a haemostatically active lamentous network. Thromb Haemost. 2007;97(4):514-526. 9. Savage B, Almus-Jacobs F, Ruggeri ZM. Specic synergy of multiple substrate-receptor interactions in platelet thrombus formation under ow. Cell. 1998;94(5):657-666. 10. Lopez JA, Andrews RK, Afshar-Kharghan V, Berndt MC. Bernard-Soulier syndrome. Blood. 1998;91(12):4397-4418. 11. Moroi M, Jung SM, Shinmyozu K, Tomiyama Y, Ordinas A, Diaz-Ricart M. Analysis of platelet adhesion to a collagencoated surface under ow conditions: the involvement of glycoprotein VI in the platelet adhesion. Blood. 1996;88(6): 2081-2092. 12. Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003;102(2):449-461. 13. Santoro SA. Identication of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986;46(6):913-920. 14. Ruggeri ZM, Bader R, de Marco L. Glanzmann thrombasthe-

22.

23.

24.

25.

26.

27.

28.

29. 30. 31.

32.

33.

34.

nia: decient binding of von Willebrand factor to thrombinstimulated platelets. Proc Natl Acad Sci U S A. 1982;79(19): 6038-6041. Hantgan RR. Fibrin protobril and brinogen binding to ADP-stimulated platelets: evidence for a common mechanism. Biochim Biophys Acta. 1988;968(1):24-35. Bennett JS, Vilaire G. Exposure of platelet brinogen receptors by ADP and epinephrine. J Clin Invest. 1979;64(5):1393-1401. Ni H, Denis CV, Subbarao S, et al. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and brinogen. J Clin Invest. 2000;106(3):385392. Jackson SP. The growing complexity of platelet aggregation. Blood. 2007;109(12):5087-5095. George JN, Caen JP, Nurden AT. Glanzmanns thrombasthenia: the spectrum of clinical disease. Blood. 1990;75(7):13831395. Quinn MJ, Plow EF, Topol EJ. Platelet glycoprotein IIb/IIIa inhibitors: recognition of a two-edged sword? Circulation. 2002;106(3):379-385. Boersma E, Harrington RA, Moliterno DJ, et al. Platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes: a meta-analysis of all major randomised clinical trials. Lancet. 2002;359(9302):189-198. Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADPinduced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem. 1998;273(4):2030-2034. Hollopeter G, Jantzen HM, Vincent D, et al. Identication of the platelet ADP receptor targeted by antithrombotic drugs. Nature. 2001;409(6817):202-207. Huang JS, Ramamurthy SK, Lin X, Le Breton GC. Cell signalling through thromboxane A2 receptors. Cell Signal. 2004;16(5):521-533. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324(7329):71-86. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee. Lancet. 1996;348(9038):1329-1339. Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov. 2010;9(2):154169. Patrono C, Baigent C, Hirsh J, Roth G. Antiplatelet drugs: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Ed). Chest. 2008;133(6 suppl):199S233S. Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A. 1999;96(20):11023-11027. Persson E, Olsen OH. Current status on tissue factor activation of factor VIIa. Thromb Res. 2010;125 Suppl 1:S11-12. Renne T, Pozgajova M, Gruner S, et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med. 2005;202(2):271-281. Salomon O, Steinberg DM, Koren-Morag N, Tanne D, Seligsohn U. Reduced incidence of ischemic stroke in patients with severe factor XI deciency. Blood. 2008;111(8):4113-4117. Rosen ED, Gailani D, Castellino FJ. FXI is essential for thrombus formation following FeCl3-induced injury of the carotid artery in the mouse. Thromb Haemost. 2002;87(4):774776. Zhang H, Lowenberg EC, Crosby JR, et al. Inhibition of the

Hematology 2011

59

35.

36.

37. 38.

39. 40. 41.

42. 43.

44.

45.

46.

47.

48. 49. 50. 51.

52.

53.

54.

55.

intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk. Blood. 2010;116(22):4684-4692. Caen J, Wu Q. Hageman factor, platelets and polyphosphates: early history and recent connection. J Thromb Haemost. 2010;8(8):1670-1674. Muller F, Mutch NJ, Schenk WA, et al. Platelet polyphosphates are proinammatory and procoagulant mediators in vivo. Cell. 2009;139(6):1143-1156. Daleke DL. Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res. 2003;44(2):233-242. Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys. 2010;39:407-427. Bevers EM, Williamson PL. Phospholipid scramblase: an update. FEBS Lett. 2010;584(13):2724-2730. Weiss HJ, Vicic WJ, Lages BA, Rogers J. Isolated deciency of platelet procoagulant activity. Am J Med. 1979;67(2):206-213. Suzuki J, Umeda M, Sims PJ, Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 2010; 468(7325):834-838. Jackson SP, Schoenwaelder SM. Procoagulant platelets: are they necrotic? Blood. 2010;116(12):2011-2018. Schoenwaelder SM, Yuan Y, Josefsson EC, et al. Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function. Blood. 2009;114(3):663-666. Kulkarni S, Woollard KJ, Thomas S, Oxley D, Jackson SP. Conversion of platelets from a proaggregatory to a proinammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading. Blood. 2007;110(6):18791886. Borissoff JI, Spronk HM, ten Cate H. The hemostatic system as a modulator of atherosclerosis. N Engl J Med. 2011;364(18): 1746-1760. Jin RC, Voetsch B, Loscalzo J. Endogenous mechanisms of inhibition of platelet function. Microcirculation. 2005;12(3):247258. Gawaz M, Neumann FJ, Ott I, Schiessler A, Schomig A. Platelet function in acute myocardial infarction treated with direct angioplasty. Circulation. 1996;93(2):229-237. Gawaz M, Langer H, May AE. Platelets in inammation and atherogenesis. J Clin Invest. 2005;115(12):3378-3384. Libby P, Ridker PM, Maseri A. Inammation and atherosclerosis. Circulation. 2002;105(9):1135-1143. Wagner DD, Frenette PS. The vessel wall and its interactions. Blood. 2008;111(11):5271-5281. Frenette PS, Denis CV, Weiss L, et al. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med. 2000;191(8): 1413-1422. Romo GM, Dong JF, Schade AJ, et al. The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin. J Exp Med. 1999;190(6):803-814. Gawaz M, Neumann FJ, Dickfeld T, et al. Vitronectin receptor (alpha(v)beta3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation. 1997;96(6):1809-1818. Massberg S, Enders G, Matos FC, et al. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood. 1999;94(11):38293838. Langer HF, Gawaz M. Platelet-vessel wall interactions in atherosclerotic disease. Thromb Haemost. 2008;99(3):480-486.

56. Dav G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med. 2007;357(24):2482-2494. 57. Massberg S, Brand K, Gruner S, et al. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002;196(7):887-896. 58. Belton OA, Duffy A, Toomey S, Fitzgerald DJ. Cyclooxygenase isoforms and platelet vessel wall interactions in the apolipoprotein E knockout mouse model of atherosclerosis. Circulation. 2003;108(24):3017-3023. 59. Hawrylowicz CM, Howells GL, Feldmann M. Platelet-derived interleukin 1 induces human endothelial adhesion molecule expression and cytokine production. J Exp Med. 1991;174(4): 785-790. 60. Kaplanski G, Farnarier C, Kaplanski S, et al. Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism. Blood. 1994;84(12):4242-4248. 61. Lindemann S, Tolley ND, Dixon DA, et al. Activated platelets mediate inammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol. 2001;154(3):485-490. 62. Gawaz M, Brand K, Dickfeld T, et al. Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis. Atherosclerosis. 2000; 148(1):75-85. 63. Gawaz M, Neumann FJ, Dickfeld T, et al. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. Circulation. 1998;98(12):1164-1171. 64. Antoniades C, Bakogiannis C, Tousoulis D, Antonopoulos AS, Stefanadis C. The CD40/CD40 ligand system: linking inammation with atherothrombosis. J Am Coll Cardiol. 2009;54(8): 669-677. 65. Kroczek RA, Graf D, Brugnoni D, et al. Defective expression of CD40 ligand on T cells causes X-linked immunodeciency with hyper-IgM (HIGM1). Immunol Rev. 1994;138:39-59. 66. Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inammatory reaction of endothelial cells. Nature. 1998;391(6667):591-594. 67. Heeschen C, Dimmeler S, Hamm CW, et al. Soluble CD40 ligand in acute coronary syndromes. N Engl J Med. 2003; 348(12):1104-1111. 68. Schonbeck U, Libby P. The CD40/CD154 receptor/ligand dyad. Cell Mol Life Sci. 2001;58(1):4-43. 69. Semple JW, Italiano JE, Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264-274. 70. Schonbeck U, Libby P. CD40 signaling and plaque instability. Circulation Research. 2001;89(12):1092-1103. 71. Ross R. Atherosclerosisan inammatory disease. N Engl J Med. 1999;340(2):115-126. 72. McEver RP, Cummings RD. Perspectives series: cell adhesion in vascular biology. Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest. 1997;100(3):485-491. 73. Simon DI, Chen Z, Xu H, et al. Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/ CD18). J Exp Med. 2000;192(2):193-204. 74. Diacovo TG, deFougerolles AR, Bainton DF, Springer TA. A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2. J Clin Invest. 1994;94(3):1243-1251. 75. Santoso S, Sachs UJ, Kroll H, et al. The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1. J Exp Med. 2002;196(5):679691. 76. Weber C, Springer TA. Neutrophil accumulation on activated,

60

American Society of Hematology

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

surface-adherent platelets in ow is mediated by interaction of Mac-1 with brinogen bound to alphaIIbbeta3 and stimulated by platelet-activating factor. J Clin Invest. 1997;100(8):20852093. Koenen RR, Weber C. Platelet-derived chemokines in vascular remodeling and atherosclerosis. Semin Thromb Hemost. 2010; 36(2):163-169. Gleissner CA, von Hundelshausen P, Ley K. Platelet chemokines in vascular disease. Arterioscler Thromb Vasc Biol. 2008;28(11):1920-1927. Brandt E, Van Damme J, Flad HD. Neutrophils can generate their activator neutrophil-activating peptide 2 by proteolytic cleavage of platelet-derived connective tissue-activating peptide III. Cytokine. 1991;3(4):311-321. von Hundelshausen P, Koenen RR, Weber C. Platelet-mediated enhancement of leukocyte adhesion. Microcirculation. 2009; 16(1):84-96. Brandt E, Ludwig A, Petersen F, Flad HD. Platelet-derived CXC chemokines: old players in new games. Immunol Rev. 2000;177:204-216. Koenen RR, von Hundelshausen P, Nesmelova IV, et al. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nature medicine. 2009;15(1):97-103. von Hundelshausen P, Weber KS, Huo Y, et al. RANTES deposition by platelets triggers monocyte arrest on inamed and atherosclerotic endothelium. Circulation. 2001;103(13): 1772-1777. Baltus T, Weber KS, Johnson Z, Proudfoot AE, Weber C. Oligomerization of RANTES is required for CCR1-mediated arrest but not CCR5-mediated transmigration of leukocytes on inamed endothelium. Blood. 2003;102(6):1985-1988. Weyrich AS, Elstad MR, McEver RP, et al. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest. 1996;97(6):1525-1534. Mause SF, von Hundelshausen P, Zernecke A, Koenen RR, Weber C. Platelet microparticles: a transcellular delivery system for RANTES promoting monocyte recruitment on endothelium. Arterioscler Thromb Vasc Biol. 2005;25(7):1512-1518.

87. Smyth SS, McEver RP, Weyrich AS, et al. Platelet functions beyond hemostasis. J Thromb Haemost. 2009;7(11):17591766. 88. Dixon DA, Tolley ND, Bemis-Standoli K, et al. Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling. J Clin Invest. 2006;116(10):2727-2738. 89. Nesbitt WS, Mangin P, Salem HH, Jackson SP. The impact of blood rheology on the molecular and cellular events underlying arterial thrombosis. J Mol Med. 2006;84(12):989-995. 90. Lehoux S, Tedgui A. Cellular mechanics and gene expression in blood vessels. J Biomech. 2003;36(5):631-643. 91. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6(1):16-26. 92. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75(3):519-560. 93. Lawrence MB, Kansas GS, Kunkel EJ, Ley K. Threshold levels of uid shear promote leukocyte adhesion through selectins (CD62L,P,E). J Cell Biol. 1997;136(3):717-727. 94. Woollard KJ, Kling D, Kulkarni S, Dart AM, Jackson S, Chin-Dusting J. Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion. Circ Res. 2006;98(1):149-156. 95. Skilbeck CA, Walker PG, David T, Nash GB. Disturbed ow promotes deposition of leucocytes from owing whole blood in a model of a damaged vessel wall. Br J Haematol. 2004;126(3): 418-427. 96. Goto S, Tamura N, Handa S. Effects of adenosine 5 diphosphate (ADP) receptor blockade on platelet aggregation under ow. Blood. 2002;99(12):4644-4645; author reply 46454646. 97. Barstad RM, Orvim U, Hamers MJ, Tjonnfjord GE, Brosstad FR, Sakariassen KS. Reduced effect of aspirin on thrombus formation at high shear and disturbed laminar blood ow. Thromb Haemost. 1996;75(5):827-832. 98. Nesbitt WS, Westein E, Tovar-Lopez FJ, et al. A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med. 2009;15(6):665-673.

Hematology 2011

61