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(9103-S)

Platelets as Fully Licensed Immune Cells

October 6, 2012 10:30 AM - 12:00 PM

Event Faculty List

Event Title: 9103-S: Platelets as Fully Licensed Immune CellsEvent Date: Saturday, October 6, 2012 Event Time: 10:30 AM to 12:00 PM

Director/Moderator/Speaker Olivier Garraud, MD PhD Director, the Auvergne-Loire Regional Blood Center Etablissement Francais du Sang [email protected] Disclosures: No

Speaker Eric Boilard, PhD Assistant professor Laval University, Rheumatology Immunology Research Center [email protected] Disclosures: No

Speaker Simon Panzer, MD Professor Medical University Vienna, AUSTRIA [email protected] Disclosures: No

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TRANSFUSION: PLATELETS CAN SENSE “DANGER” SIGNATURES AND SIGNAL “OTHER” IMMUNE CELLS TO RESPOND BY INFLAMMATION

Prof. Olivier GARRAUD MD PhD_Head, the Regional Auvergne-Loire Blood Center of the French National Blood Service (EFS)_Faculty of Medicine of Saint-Etienne, University of Lyon, France

Garraud O, Cognasse F, Hamzeh-Cognasse H et al. Saint-Etienne, France — AABB Boston 2012

Blood collection: Platelet Component (PC)preparation

• Blood collection for making a PC (aphaeresis, buffy coat) inflicts cell lesions and some degree of activation (CD62P, sCD62P, CD63, cytokine production/release) mechanical stress, anticoagulant

• Storage in view of constituting a PC inventory (≤5 d) also inflicts platelet lesions(activation, cytokine production/release, emission of microparticles MPs etc.) contacts with plastics, gas exchanges, floatation in medium… chemical stress, ageing

-Cognasse F et al., Transfusion, 2006;46:1184-1189-Cognasse F et al., Transfusion, 2008a;48:809-813-Chavarin P et al., Vox Sang, 2011:100:247-249-Nguyen KA et al., Blood Transfus, 2012:July 12:1-2Further work either submitted or in preparation…


Blood Transfusion: PC linked-hazards_1.Frequency

PCs: ~10% of deliveries;39.4% of incidents/accidents

Data from the French Hemovigilance 2011; ANSM 2012


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Blood Transfusion: PC linked-hazards_2.Severity


B cells

Plateletssupernatantor lysate

Activation:IL-6 release

ATR d5 (n=1) and d7 (n=3)Control d5 (n=10 and d7 (n=10)

Centrifugation

Plateletssupernatant

Plateletslysate

CD40L soluble

Supernatant Pellet

ATR +++ -

Control + ++

B cells: IL-6

PlateletSn

PelletSn

ATR +++ -

Ctl + ++

CD40-blocking antibodiessubstantiallyabrogated IL-6 secretion.

sCD40L…

CD40

PC transfusion linked hazards: Involvement of inflammatory cytokines

30 ART cases29 w/. sCD40L + IL-27 + Ox40L1 w/. sCD40L + IL-27

H. Hamzeh-Cognasse et al., submitted


Platelets: Origin of the main molecules with relevance to transfusion_The 3 main sources of pltoriginating molecules:

1.Megakaryocytes 2.Absorbed from the environment (plasma)

3.Synthesized de novo (Weyrich et al.)

_The 3 main locations of pltoriginating molecules:

1.The membrane 2.The granules 3.The dense () granules

_The 3 main processes for gaining access to plt originating molecules:

1.Shedding(membrane)

2.Direct secretion(, )

3.Two time secretion (=> membrane => outside)()

_The 3 main categories of pltoriginating molecules:

1.Membrane GP 2.Signalosome Phospho-Proteins

3.Docked GP and other molecules


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Platelet, Signaling and beyondGarraud O, Cognasse F, Hamzeh-Cognasse H et al. Saint-Etienne, France — AABB Boston 2012

Platelet activation with LPS (E. coli, 1 µg/ML, 30 min)(picture by Charles-Antoine Arthaud, Saint-Etienne)

Dense Granules

Lysosomes

Chemokines

Alpha Granules

ADP, ATP, Ca++, Histamine, 5 HT…

RANTES, PF4, IL-8, ENA-78, MIP1a…Cell mitogens: PDGF, EGF, TGFß, VEGF…Hemostatic factors: Fibrinogen, PAI1, Plasminogen…Adhesion Proteins: Thrombospondin, vWF…Critical Ligands: GPVI, P-Selectin, CD40L/CD154

Platelets and Pathogen Recognition Receptors (PRR): the Toll like Receptors (TLRs)


Platelet-TLR4 are functional and, upon LPS engagement, secrete or dock (differentially) an array of cytokines and the like


Platelet1

Platelet2

TLR4LPS

1LPS

2

Signalisation 1MyD88, predominant

Signalisation 2TRIF, predominant

NFB

Cytokine SecretionProfile 1

Cytokine SecretionProfile 2

Activationsignalisation

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Platelets have the molecular machinery for signaling through TLR4


Toll-like receptor 4 (TLR4) expression on platelet membranes. (A) TLR4 was detected by flow cytometry after gating on CD41+ cells. Platelets were unstimulated or stimulated (30 min, RT) with TRAP. One representative experiment is shown (n=10). (B) The mean percentage of CD41+ platelets also positive for TLR4, as determined by flow cytometry (mean ± SD from 10 independent experiments).

A

59% 70%

TLR4 expression gated on unstimulated platelet CD41+

TLR4 expression gated on TRAP stimulated platelet CD41+

59% 70%

TLR4 expression gated on unstimulated platelet CD41+

TLR4 expression gated on TRAP stimulated platelet CD41+

1%

Isotype control

TLR4

Human platelets can discriminate between various bacterial LPS isoforms via TLR4 signaling and differential cytokines secretionBerthetJ et al. , 2012 Clinical Immunology (in press)


Platelets + LPS

Supernatant

PBMCs

Supernatant

TLR2 Signaling in plateletsGarraud O, Cognasse F, Hamzeh-Cognasse H et al. Saint-Etienne, France — AABB Boston 2012

Human platelet NF-kappa B links TLR2 and PAR1 to cytokine secretion

Pauline Damien1, FabriceCognasse1, 2, Bernard Payrastre3, Sherry L. Spinelli4, Neil Blumberg4, Richard P. Phipps4, Archibald McNicol 5, Bruno Pozzetto1, Olivier Garraud1, 2 and Hind Hamzeh-Cognasse1

Submitted for publication

*# *#

*# *#

*#

*# *#

*

*#

*# *#

*#

A

B

TRAP

Pam3CSK4

Both TRAP and TLR2-ligand triggering involve NFκB signaling but with different kinetics and intensity.

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Platelet Serotonin, sCD62p, RANTES and sCD40L released by platelets stimulated by TRAP or Pam3CSK4, with (w) or without (w/o) anti-human TLR2 blocking MoAb.

Serotonin


Serotonin, sCD62p, RANTES and sCD40L secretion levels from platelets stimulated by Pam3CSK4 (A, C, E and G) or TRAP (B, D, F and H) with (w) or without (w/o) the inhibitor of NF-κB (BAY 11-7082).

While TRAP induces the release of dense and α-granule content, TLR2-ligand selectively and specificallypromotes the release of α-granule content.

_Platelets: Other Sensors of Danger and Receptors

After: Flaujac C et al., CellMol Life Sci, 2010;67:545-556 (Review)

Platelet

FcR()

CR

PRR

TLR (ligands = PAMP)

Defensins (ligands =DAMP tissue damageand/or repair)


a2b1

CR2

CCR1CCR3CCR4CXCR4

DC-SIGN

GPVI

a2b3CARCLEC2

VP4 (Rotavirus)

EBV

HIVHIV, Lentivirus

HCV

AdenovirusHantavirus

CoxsackieHIV

Other viruses, bacteriaeOther types of microbes

20/09/2012

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Hypothesis: Blood processing for constituting PCs may harm cells and present as a “danger”


Platelets engage mutual interactions with leukocytes: relevance in transfusion


Platelet binding to PBMC increases upon platelet storagePlatelets bound to B cells, monocytes and T cells induce their activation.

Platelets and B lymphocytes were mutually activated; after a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, IgG3, but not IgG4, IgA, IgM

The release of platelet-derived nucleotidic molecules is inhibited upon binding of platelets to DC, thus limiting DC activation and promoting a tolerogenic phenotype

Physiopathology of PC linked-inflammation:_Acute Transfusion Reactions (ATRs)• 1. Non Hemolytic Febrile Transfusion Reactions (NHFTRs)

Predictive value of cytokine secretion in the platelet component in ATRs

Observations in 65 non-TRALI cases (grades 3 & 4; accountability 2 and over)

Nguyen KA el al, in preparation for publication


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• 2. Transfusion Related Acute Lung Injury (TRALI)

Physiopathology of PC linked-inflammation:_Acute Transfusion Reactions (ATRs)

Silliman C, Blumberg N, Phipps R, et al. Blood (2006)

Blood Component

Fresh plasma (healthy)

Platelets from whole blood

Platelets collected by apheresis

CD40 Ligand levels (ng/ml)in blood products

No TRALI TRALI

0.01 ± 0.003 N/A

7.9 ± 1.114.0 ± 3.0

11.8±0.82 44.7 ± 4.5

Acute pulmonary insufficiency induced by transfusion and involving neutrophil damage to endothelium; Cohort study -

534 platelet transfusions

Blumberg/Heal/Phipps Transfusion 2006;46:1813


• Platelets activate DCs and interfere with T cells, B cells, monocytes etc.

• Platelets would be able to function as APCs (Chapman LM, J Immunol2012;189:916-923), though debatable

• Physiological inflammation is instrumental in assisting APC function (cartoon)

• Transfusion represents an inflammatory situation (Transfus ClinBiol, special issue, 2012;19:81-138)

• HLA Ags, HPA Ags; ITP; FNAIT: role of cytokines during pregnancy?

• Do transfused platelets favor APC of their own Ags?

Physiopathology of PC linked-inflammation:_Speculation: A role in Allo-Immunization

Capture

SynthesisDegradation

CaptureSignalingActivation

SynthesisDegradation

-Cytokines (pro-inflamm.)-MHC molecules

s

-Peptides (to be presented)-Debris (inflammatory)

Antigen Presenting Cell

T cell

Th1

Th1

Th1

B

Mo

Inflammatory cytokines and products

Th2

B


Conclusions and perspectives


Therapeutic principle (Platelets)

+Cytokines, Chemokines, BiologicalResponse Modifiers,&Residual plasma, anticoagulant, etc.

Donor Plateletsin the Recipient

Circulation Arborescence

RecipientImmune Cell

Foreign ¢ (platelets)

Stressed Platelets

_Platelet DAMP_Immune ¢ Sensor/Defensin

Infused Donor Platelets maysignal Sensors in the Recipient Inflammation

(+)?: healing

(-): ATR + immunization

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In summary:• Platelets are fully licensed immune cells because they have the

main 4 properties that entitle them for this characteristics (Garraud,

Hamzeh-Cognasse, Cognasse, Pozzetto, Cavaillon, Weyrich & Semple: Review submitted for publication).• Expression of non-mutated receptors => bind pathogen or pathogen

derived structures• Ability to ingest pathogens• Capacity of secreting cytokines, chemokines, inflammation mediators• Capacity of activating a signaling machinery in a polarized manner

• It has been suggested that they present Ag• They interact w/ innate immune cells from the environment and

w/ adaptive immune cells, and alter their physiology• They are involved potentially in physiological inflammation and

certainly in pathological inflammation• More?


Main collaborations

• University of Louvain, Clinics of Mont‐Godinne, Belgium

• University of Winnipeg, CN

• Etablissement Français du Sang (several other regions)

• Institut National de la Transfusion Sanguine

• CHU de Saint‐Etienne

• Université de Lyon

• INSERM (Lyon, Paris, Toulouse)

• Insitut Cochin, INSERM & CNRS, Paris

• University of Monastir and University Hospitals and Blood Banks in of Sousse, Monastir and Tunis; & the Pasteur Institute in Tunis.

• King Saudi University, Saudi Arabia; the Assiut University, Egypt

• Special thanks to advisers: Pr Andy Weyrich, Salt Lake City, USA; Pr John Semple, Toronto, CN; Prs Neil Blumberand Rick Phipps, and Dr S. Spinelli, Rochester‐NY, USA; Pr Larry Corash, University of California, San Francisco, USA; Pr B. Payrastre, CNRS, Toulouse, FR

Main Fundings

• Etablissement Français du sang

• Université Jean‐Monnet de Saint‐Etienne

• Association Recherche Transfusion

• Association les Amis de Rémi

• Région Rhône‐Alpes (C‐MIRA) – partnershipwith the University of Monastir in Tunisia


Merci pour votre attentionFabrice COGNASSE,

PhD

Sandrine LARADI, Pharm D, PhDHind HAMZEH-COGNASSE, PhD

Patricia CHAVARIN, MD

Olivier GARRAUDMD, PhD, Prof.Kim A NGUYEN

MD, PhD Student

Missing: Vincent BOST, MD (clinical investigations) & Antoine PRIGENT (MD, PhD Student)

ChakerALOUI,PhD Student)

PaulineDamien, PhD student

Pr. Bruno POZZETTOHead of the Univ. group

20/09/2012

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Platelet functions beyond hemostasis

Simon Panzer, MD

Medical University Vienna, [email protected]

Disclosure

Unfortunately, none

platelet -activation, -function

Alan Michelson 2005

20/09/2012

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subendothelial matrix

Ib IbIa/IIa

IIb/IIIa*Ib

Ia/IIaVI/Fc

IIb/IIIa*

primary adhesion

firm adhesion

activation

vWF

endothelial cell

collagen

Ib IbIb

fibrinogen

endothelial cell

blood flow

Kehrel BE, with permssion

platelets’ hemostasis

subendothelial matrixvWF

endothelial cells

endothelial cells

collagen

IIb/IIIa*Ib

fibrinogen

P-selectin TSP

recruitment

ADP Thromboxan A2

MMP

ADPadhesion proteins growth factors

spreadinggranule

secretion

IIb/IIIa*

Kehrel BE, with permssion

thrombin

platelets’ hemostasis

Platelets in Love

20/09/2012

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PMNL

subendothelial matrix

endothelial cell‐

endothelial cell

collagen

monocyte

aggregation + recruitment of leukocytes Kehrel BE, with permssion

the clot

Platelets as targets of an immune response

(Auto)Immune Thrombocytopenia

antibody‐mediated, T cell mediated, reactive oxygen species

Allo‐Immune: NAIT, Refractoriness to platelet transfusions, Post‐transfusion Purpura

antibody‐mediated

platelets contain what you need for life, or will find the relevant source

you

Platelets store bioactive mediators

Platelets contain mRNA to generate their own proteins

20/09/2012

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beyond hemostasis

cancer proliferation

viral spreading, promote cytotoxic T cells

host defense against microorganisms

infection/inflammation ‐ thromboembolism

Drawings from Bizzozero‘s original paper (1882)

„every time when a vascular wall is damaged ... arrest of whiteblood corpuscles represents a secondary phenomenon and may, perhaps, be caused by increasedadhesive properties of bloodplatelets whereby these cellsreact with white blood corpuscleswhich have been brought intocontact with them by bloodcirculation“

Bizzozero (1882)

key: platelets adhering to leukocytes

1963

Bolton F G & Boyd JPlatelet adherence to polymorphs. Br. Med. J. 2, 747 (1963)

Crome P E & Barkhan PPlatelet adherence to polymorphs. Br. Med. J. 2, 871 (1963)

Field E J & McLeod IPlatelet adherence to polymorphs. Br. Med. J. 2, 388 (1963)

„We cannot relate this phenomenon to anyfunctional abnormality of the blood nor to thepatient‘s symptons, but we think the appearance issingular enough to be worth reporting.“

20/09/2012

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putative adhesion molecules involved in platelet adhesion to leucocytes

platelets leukocytes

P-selectin PSGL-1

GPIIb/IIIa, GPIb, Jam-3 Mac-1

CD40L CD40

ICAM-2 LFA-1

CD47 CD36

CD45 CD45CD45

CD45

CD61

CD61

CD61

CD61

Granulocytes Monocytes Lymphocytes Platelets

w/o agonistplatelets adhering to leukocytes

resting activated

Human platelet antigen-1a antibodies induce the release of the chemokine RANTES from human platelets

Dettke M et al, Vox Sang 2001; 81:199

the release of RANTES is FcII dependent

RANTES from platelets, can bind in flamed endothelium, forming a bridge between mononuclear cells and the vascular wall. RANTES also induces rapid intracellular signaling events in leukocytes and, in monocytes, directly signals to gene expression pathways that control in fammation

alloantibodies induce cytokine release

Platelet activation can result in release of multiple and diverse soluble mediators with pleiotropic functions in inflammation

20/09/2012

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adhesion molecules involved in plateletadhesion to leucocytes

platelets ligands leucocytes

P-selectin - PSGL-1

GPIIb/IIIa fibrinogen Mac-1 (CD11b/CD18)

or CD11c/CD18GPIIb/IIIa fibronectin

vitronectinthrombospondin

Mac-1 (CD11b/CD18)

CD40L - CD40

ICAM-2 - LFA-1 (CD11a/CD18)

GPIbα high molecular weight kininogen

Mac-1 (CD11b/CD18)

Jam-3 - Mac-1 (CD11b/CD18)

CD47 thrombospondin CD36

New Links Between Inflammation and Thrombosis

Wagner DD, ATVB 2005;25:1321

.

the interplay of platelets and leukocytes in thrombosis and inflammation

‐ platelet P‐selectin contributes significantly to atherosclerotic lesion growth

‐ platelet P‐selctin activates endothelial cells inducing leukocyte rolling

‐ P‐selectin regulates fibrin deposition

‐ P‐selectin induces microparticles from monocytes (tissue factor)

‐ platelet P‐selectin capture leukocyte microparticles (via PSGL‐1) to thrombi (TF)

soluble CD40L (CD154) comes from platelets

regulation of adaptive immune resonse

supports B cell differentiation, Ig‐switchaugment CD8+ T cell response

platelet CD40L promotes DC maturationreduction of proinflammatory cytokines

Sprague DL et al, Immunol Res 2007;39:185

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Platelet components associated with acute transfusion reactions: the role of platelet-derived

soluble CD40 ligandCognasse F et al, Blood 2008; 112:4779

sCD40L in platelet concentrates (PCs) is associated with clinical Acute Transfusion Reaction through B‐cell responses as an indication of pathophysiologic function. There is a sustained role for PC‐derived sCD40L.

Toll-like receptors (TLR)

pattern recognition receptors (PRR): recognize molecules broadly shared by pathogens distinguishable from host molecules, referred to as pathogen-associated molecular patterns (PAMPs)

TLR on human platelets

Aslam R et al Platelets 2004; 15: 267Shiraki R et al Thromb Res 2004; 113:379Andonegui C et al Blood 2005; 106:2417Cognasse F et al Immunol Cell Biol 2005; 88:196

thrombin activated platelets increaselevels of TLR4 and TLR9

resting thrombin activated resting thrombin activated

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Platelet Toll‐like receptor expression modulates lipopolysaccharide‐induced thrombocytopenia and tumor necrosis factor‐alpha production in

vivoAslam R et al, Blood 2006;107:637

bridging infection with thrombosis:

Thrombin and LPS induce TLR 4 and TLR 9

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic bloodClark SR et al, Nature Medicine 2007; 13: 463

Platelets and the immune continuumSemple JW et al Nature Reviews 2011; 11: 265

Bacteria bind to platelet TLR4 and are killed directly or by “trapping” provide professional phagocytosisPlatelet TLR4 presents LPS to neutrophils promoting their activation

Lipopolysaccharide from enterohemorrhagicEscherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Stahl AL et al Blood 2006; 108: 167

Platelet TLR4 binds only to LPS of E.colienterohaemorrhagica if associated with haemolyticuremic syndrome (HUS), but not not if infection is not leading to HUS. This mechanism may explain platelet consumption in HUS

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Human neutrophil alpha‐defensins induceformation of fibrinogen and thrombospondin‐1 amyloid‐like structures and activate platelets via glycoprotein IIb/IIIa.HORN M et al, J Thromb Haemost 2012; 10: 647

Defensins activate platelets and induce platelet apoptosis by formation of amyloid-like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiolisomerasesBertling et al, ATVB 2012; 32: 1979

staphylococcus aureus can induce platelet aggregationsurface-located thiol isomerases play an important role in platelet activation

staphylococcal extracellular adherence protein induced thiol isomerase-dependent

glycoprotein IIb/IIIa activation, granule secretion, platelet aggregation

The prothrombotic features of a microbial secreted protein could explain infection-associated thrombosis

Defensins kill bacteria

platelets’ granule: thrombocidinsPlatelets kill intraerythrocytic malarial parasites and mediate survival to infectionMcMorran B J et al, Science 2009; 323: 797

platelets can kill parasites in vitro and in miceplatelet‐deficient mice are more likely to die from malaria than mice with normal platelet countsa single dose of aspirin may interfere with platelets sufficiently to prevent their killing power

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selection for further readings

The evolving role of platelets in inflammationWeyrich AS, J Thromb Haemost 2003; 1:1897

New links between inflammation and thrombosisWagner DD, ATVB 2005; 25:1321

Platelets and innate immunitySemple JW, Cell Mol Life Science 2010; 67:499

Platelets and the immune continuumSemple JW, Nature Rev Immunol 2011; 11:264

Bidirectional crosstalk between platelets and monocytes initiated by Toll-like receptor: An important step in the early defense against fungal infections?Bruserud O, Platelets 2012; PMID: 22646762

PlateletsMichelson AD Elsevier, second edition 2007

summary of platelets’ functions

• ensure haemostasis

‐ clot formation

‐ initiation/interaction of plasma coagulation

‐ secure the vascular integrity

‐ vessel repair

• surveillance‐ Inflammation

‐ bridging innate and adaptive immune response

‐ Fully licensed immune cells

inspired by work and discussions

John Semple, Toronto, Canada

Andy Weyrich, Salt Lake City, USA

Beate Kehrel, Muenster, Germany

Olivier Garraud, Lyon, France

Alan Michelson, Boston, USA

Platelet functions beyond hemostasis

This presentation will comprise selected topics showing platelets’ function beyond haemostasis with emphasis on the interaction of platelet with leukocytes, how platelets bridge innate and adaptive immunoresponses and platelets as a killing machinery of bacteria. The interaction between platelets with bacteria via platelets’ Toll-like receptors shall illustrate how infections can become procoagulatory.

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