Biology of monocyte interactions with the endothelium : the platelet factor - Thesis

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Biology of monocyte interactions with the endothelium : the platelet factor

da Costa Martins, P.A.

Publication date

2005

Document Version

Final published version

Link to publication

Citation for published version (APA):

da Costa Martins, P. A. (2005). Biology of monocyte interactions with the endothelium : the

platelet factor.

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Thee Biology of Monocyte

Interactionss with the Endothelium:

thee Platelet Factor

thee Platelet Factor

Thee studies here described were performed at the Department of Experimental Immunohematology,, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medicall Centre, University of Amsterdam, the Netherlands

Thee Biology of Monocyte Interactions with the Endothelium: the platelet factor

ISBNN 90-9019886-5 Authorr Paula da Costa Martins

Coverr and layout Paula da Costa Martins

Printt PrintPartners Ipskamp, Enschede, the Netherlands

©© 2005 by Paula da Costa Martins, Amsterdam

Thee printing of this thesis was financially supported by J.E. Jurriaanse Stichting; by Sanquin Research att CLB; by the Academical Medical Centre, University of Amsterdam; by Dr. Ir. J.H. van der Laar Stichtingg and the company Sanquin CLB Marketing Reagentia.

endothelium m

the platelet factor

-ACADEMISCHH PROEFSCHRIFT

terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus

prof.. mr. P.F. van der Heijden

tenn overstaan van een door het college voor promoties ingestelde commissie,, in het openbaar te verdedigen in de Aula der Universiteit

op p

woensdagg 26 oktober 2005, te 12.00 uur

door r

Paulaa Alexandra da Costa Martins

Promotiee commissie:

Promotor:: Prof. dr. D. Roos Co-promotor:: Dr. J.J. Zwaginga

Overigee leden: Prof. dr. C.G. Figdor Prof.. dr. C. Ince

Prof.. dr. L. Koenderman Prof.. dr. M.H.J. van Oers Dr.. E.S.G. Stroes

Faculteitt der Geneeskunde

Thee study described in this thesis was supported by a grant of the Netherlands Heart Foundationn (NHF-1999B059).

Financiall support by the Netherlands Heart Foundation for the publication of this thesiss is gratefully acknowledged.

ee com vento fiz a vela que me leva souu pescador de coisas belas, de emocöes souu a mare que sempre sobe e nao sossega

Souu das pessoas que me querem e que amo vivoo com elas por saber quanto Ihes quero aa minha casa é uma ilha é uma pedra quee me entregaram num abraco sincero

Souu doutras coisas souu de pensar que a grandeza esta no homem porquee é o homem o mais lindo continente tantoo me faz que a terra seja longa ou curta tranco-mee aqui por ser humano e por ser gente..."

Fernandoo Tordo in Anticiclone, 1984

Chapterr 1 Introductionn and aim of study 11 1

Chapterr 2 Platelet-monocyte complexes support monocyte

adhesionn to endothelium by enhancing secondary tetheringg and cluster formation

AtheriosclerAtherioscler Thromb Vase Biol (2004) 24:193-199

37 7

Chapterr 3 P-selectin Glycoprotein Ligand-1 (PSGL-1) is expressed

onn endothelial cells and mediates monocyte adhesion too activated endothelium

SubmittedSubmitted for publication

55 5

Chapterr 4 Platelet binding to monocytes increases the adhesive

propertiess of monocytes by upregulating the expression andd functionality of fr and (32 integrins

SubmittedSubmitted for publication

77 7

Chapterr 5 LDL-receptor - related protein regulates (32 integrin

-mediatedd leukocyte adhesion

BloodBlood (2005) 105:170-177

97 7

Chapterr 6 DC-SIGN mediates adhesion and rolling of dendritic cells

onn primary human umbilical vein endothelial cells through LewisYY antigen expressed on ICAM-2

ManuscriptManuscript in preparation

119 9

Chapterr 7 Discussion, concluding remarks and future perspectives 139 9

Nederlandsee samenvatting/Sumario em português Acknowledgements/Dankwoord/Agradecimentos s

CurriculumCurriculum vitae

155 5 165 5 169 9

Abbreviations s ADP P ADPase e ApoE E BSA A COX X CHO O CVD D DNA A dNTP P DMEM DMEM DC C DC-SIGN N ECM M EC C ERK1.2 2 EDTA A ELISA A FACS S FMVEC C FITC C GlyCAM-1 1 GP P GST-1,-2 2 GAPDH H GDP P GFP P HUVEC C HSA A HEPES S HRP P HLA-A, , HIT T ICAM-1,, -2 IL-1,-8 8 IFN-Y Y kDa a LFA-1 1 LDL L LRP P LeY,, (s) Le x , Lea MadCAM-1 1 MAPK K MEK1 1 MCP-1 1 MoAbs s MACS S PMNs s PMPs s -B,-C C Leb b adenosinee 5'-diphosphate

adenosinee 5'-diphosphate synthase apolipoproteinn E

bovinee serum albumin cyclooxygenase e chinesee hamster ovary cardiovascularr disease deoxyribonucleicc acid deoxynucleotides s

Dulbecco'ss modified Eagle medium dendriticc cell

DC-specificc ICAM-3 grabbing non-integrin extracellularr matrix

endotheliall cell

extracellularr signal-regulated kinases 1 and 2 ethylenediaminetetraaceticc acid

enzyme-linkedd immunosorbent assay fluorescencee activated cell sorter foreskinn microvascular endothelial cell fluoresceinn isothiocyanate

glycosylation-dependentt cell adhesion molecule-1 glycoprotein n

glutathionee S-transferase

glyceraldehydee 3-phosphodehydrogenase guanosinee diphosphate

greenn fluorescent protein

humann umbilical vein endothelial cell humann serum albumin

N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonicacid) ) horseradishh peroxidase

humann leukocyte antigen, class I heparin-inducedd thrombocytopenia Intercellularr adhesion molecule-1 and -2 interleukine-11 and -8

interferon-y y kilodalton n

lymphocytee function antigen-1 low-densityy lipoprotein

low-densityy lipoprotein receptor-related protein Lewiss antigen

mucosall vascular addressin cell adhesion molecule-1 mitogen-activatedd protein kinase

mitogen-activatedd protein kinase kinase 1 monocytee chemoattractant protein-1 monoclonall antibodies

automatedd magnetic cell sorting and separation polymorphonuclearr cells

PMC C PBS S PRP P PE E PCR R PVDF F PMA A PBMCs s PSGL-1 1 RANTES S RBC C RNA A RT-PCR R RAP P SEM M SDS-PAGE E SPR R TNF-a a TF F TRAP P TSM M uPAR R VLA-4 4 vWF F platelet-monocytee complex phosphate-bufferedd saline platelet-richh plasma phycoerythrine e

polymerasee chain reaction

polyvinylidenee fluoride (transfer membrane) phorboll 12-myristate 13-acetate

peripherall blood mononuclear cells P-selectinn glycoprotein ligand-1

regulatedd on activation normal T expressed and secreted redd blood cells

ribonucleicc acid

reversee transcriptase PCR receptor-associatedd protein standardd error of the mean

sodiumm dodecyl sulfate-polyacrylamide gel electrophoresis surfacee plasmon resonance analysis

tumorr necrosis factor a tissuee factor

tartratee resistant acid phosphatase tris-saline-magnesiumm buffer

urokinasee plasminogen activator receptor veryy late antigen 4

Chapterr 1

Thrombosiss and atherosclerosis, in relation to cardiovascular diseases, are majorr determinants of morbidity and mortality in the Western societies. Scientific research,, so far, has focused mainly on non-cellular risk factors for both processes. Forr example, deficient inhibition of coagulation and fibrinolysis have been delineated ass causes for thrombosis, and aberrant lipid and/or cholesterol metabolism received mostt attention as a cause for atherogenesis. However, these processes are decisivelyy determined by cellular responses: firstly by the magnitude or chronicity of endotheliall stimulation and, secondly, by the subsequent inflammatory and hemostaticc response to this stimulation. The responses and interactions of endotheliall cells with leukocytes and platelets are the main subject of this introduction. .

Togetherr with platelets, inflammatory cells are known to play a role in thrombosis and atherosclerosis.. In vivo adhesion and influx of phagocytes at and within the thrombus hass been object of several studies 1"3. The role of phagocytes can be advantageous ass they are considered to play a role in the reorganization of the thrombus and neededd to restore vascular integrity and new vessel formation. Moreover, the presencee of inflammatory cells at these sites, where pathogen invasion is a risk, mightt be beneficial. On the other hand, monocyte and lymphocyte infiltration can be viewed,, pathologically, as the first inflammatory events in the development of atherosclerosis.. As lipid-laden foam cells are derived from monocytes 4'5, in a more progressedd lesion, these foam cells correlate with a high risk of plaque rupture, expressionn of tissue factor and subsequent activation of the coagulation cascade. Thee current paradigm of atherosclerosis as an inflammatory process that promotes lesionn development and progression has been established throughout the years. By consideringg the importance of this inflammatory process, in this thesis we will show evidencee of strong interaction between the hemostatic and the inflammatory response,, as well as present new insights into the mechanism of recruitment of leukocytess and platelets to the inflamed vessel wall. This general introduction briefly describess the early hemostatic events after vessel wall injury and the cellular adhesionn molecules that mediate adhesion of leukocytes and/or leukocyte-platelet aggregatess under flow conditions.

Platelets/thrombii at the vessel wall induce colocalisation of leukocytes

PlateletPlatelet adhesion to injured vessel wall. Platelet function and, in particular,

plateletss capacity to adhere, to become activated, to form aggregates and to support locall initiation of coagulation, play a central role in normal hemostasis. To avoid harmfull thrombus formation, platelet activation usually only occurs at sites of vascularr damage and, more drastically, when the subendothelial tissue is exposed. If thee latter occurs in the arterial vascular bed, shear stress and red blood cell

-dependentt platelet margination will favor the formation of a thrombus consisting mainlyy of platelets (white thrombus)6"8. Due to its shear-dependent functionality, Von Willebrandd factor (vWF) plays a major role in thrombus formation as the most importantt plasma and vascular glycoprotein mediating platelet adhesion 9. In contrast,, venous thrombi mostly consist of a loosely packed network of erythrocytes andd islands of aggregated platelets reinforced/encapsulated by fibrin that can be formedd at lower shear stresses ("red thrombus"). Vessel wall injury and subsequent exposuree of the extracellular matrix (ECM) may be caused by disturbed blood flow, hypertension,, platelet- or leukocyte-released products, bacteria, endotoxins, viruses, smoking,, dietary lipids, homocystenemia, diabetes, other metabolic disorders, and stresss 9. Under these conditions, platelet-dependent hemostasis is initiated 61 (Figuree 1). Platelets readily adhere to vWF and collagen in the extracellular matrix andd become activated. Subsequently, there is an increase in adhesivity, initiated by collagenn and locally generated thrombin, and release of platelet-activating substancess enabling platelet aggregation 1112.

bloodd flow

tetheringg

thrombus

7 \\ . adhesion formation

CC -* endothelial cell

tt activated platelet

Figuree 1. Multistep adhesive interactions of platelets with vascular surfaces under flow.

Adhesionn process and molecules involved in platelet recruitment to subendothelial surfaces.

Activationn of endothelial cells, without ECM exposure, also support platelet deposition.. However, these are mostly transient and short-termed interactions leadingg to rolling of platelets over the vessel wall 13,14. In contrast, activated endotheliall cells with subsequently upregulated adhesion receptors are more adhesivee for inflammatory cells than the sites of endothelial vascular wall damage. Therefore,, the inflammatory and hemostatic responses are determined by the

conditionsconditions that disturb the integrity of the endothelial monolayer. The extreme end of thesee processes can be mechanical denudation or plaque rupture, with endothelial

Platelet-derivedd microparticles at damage sites have been shown to increase the adhesivee interactions of neutrophils, monocytes or monocytoid cells 15 with the endothelium.. This indicates that platelets and platelet-derived products at sites of vessell wall injury may not only influence thrombosis but also modulate subsequent multicellularr interactions that can be of relevance for the early stages of atherogenesis. .

LeukocyteLeukocyte adhesion to activated endothelium. Stimulation of endothelial

cellss by e.g. bacterial chemokines, leads to upregulation of adhesion molecules on thee cell surface. However, leukocytes also need to be activated before stable firm adhesionn and subsequent migration can take place. The presence of specific adhesionn receptors and leukocyte-activating chemokines act as a kind of an area codee and start a process of multistep adhesion and endothelial diapedesis of leukocytess at these sites.

MultistepMultistep paradigm of leukocyte extravasation {Figure 2). The first short-term

interactionss between leukocytes and endothelial cells are called "primary tethering". Subsequently,, the leukocytes slow down to a speed a thousand times lower than the freee flowing cells, and show a rolling interaction with the endothelium. Rolling is dependentt on a minimum of force exhibited by the blood stream, called "shear stress"16,17.. Both primary tethering and rolling of leukocytes over the endothelium are mediatedd by the selectin family of adhesion molecules (Figure 3A). E-selectin is, with aa slight delay after stimulation (e.g. by TNF-a), upregulated on activated endothelial cells.. L-selectin is constitutively present on leukocytes 18"20 and, in contrast to E-selectin,, shed from the surface when leukocytes become activated 21"24. P-selectin is storedd in Weibel-Palade bodies of EC and a-granules present in platelets 25"27. P-selectinn is expressed transiently on the surface of these cells after stimulation with variouss inflammatory stimuli e.g. histamine or thrombin on EC.

P-- and L-selectin are primarily responsible for tethering of flowing leukocytes to the endotheliumm but they also support cell rolling. E-selectin is particularly important for slowingg the rolling velocity of leukocytes after they have tethered through P- or L-selectinn 28. These slower rolling interactions increase the probability of firm adhesion, necessaryy for final extravasation of leukocytes in different ways: A. by allowing firm adhesionn via low-affinity receptor-ligand interactions and, B. by increasing the leukocyte'ss probability of binding chemokines on the endothelial cell surface29. Chemokiness will activate the leukocyte by transducing signals that intersect with thosee produced by engagement of selectin ligands 20,3 , usually leading to increased expressionn and/or increased affinity of receptors on the leukocyte membrane.

monocyte e

capturee rolling firm adhesion diapedesis

Figuree 2. Multistep model of leukocyte extravasation. At sites of inflammation, leukocytes

tether,, or form initial attachments, to vascular endothelial cells. They then roll, or move continuously withh rotational movements, on the vessel wall, until they finally arrest, spread and migrate between endotheliall cells into the surrounding tissues.

Firmm adhesion is mediated by integrins on the leukocytes that bind to their ligands on thee endothelium, belonging to the immunoglobulin superfamily (Figure 3B). On non-activated,, resting leukocytes, integrins maintain a conformationally inactive state. Uponn stimulation by chemoattractants or other stimuli, integrins change their conformation,, leading to increased affinity and/or avidity for their ligands. This processs is called inside-out signaling 31"34. Integrins, by interacting with their respectivee endothelial receptor molecules (e.g. ICAM-1, -2 or VCAM-1) mediate firm adhesionn of leukocytes to the vessel wall 35~39. Subsequently, the adhering leukocytess crawl to the site where endothelial cells are connected to each other and migratee in between two adjacent cells to the inflammatory site. Additionally, the bindingg of integrins to their ligands, by itself, leads to activation of intracellular signalingg pathways in the leukocytes and in the endothelial cells (outside-in signaling)) 40. In this way also endothelial cells are able to respond to inflammatory conditions.. For example, it has been shown that outside-in signaling by adhesion of neutrophilss to endothelial receptors increases the endothelial permeability by disorganizingg endothelial cell-to-cell adherent junctions, thus facilitating trans-endotheliall migration 41. Another possible adhesion-regulated response may be the migrationn through an endothelial cell instead of in between two adjacent endothelial cellss as shown for neutrophils 42. After passing the endothelium, the leukocytes will interactt with the subendothelial matrix and migrate to the site of inflammation 4344.

AA B

PSGL-11 ESL-1

sLex,, Lex sLex, Lex L-selectin Mac-1 LFA-1 VLA-4

P-selectinn E-selectin MadCAM ICAM-1 ICAM-2 VCAM-1 GlyCAM M

sLex x

Figuree 3. Selectins, integrins and respective ligands presented by leukocytes and endotheliall cells. A. Interactions of selectins with the respective cell-surface glycoconjugates mediate

tetheringg and rolling adhesion of leukocytes on the vessel wall. PSGL-1 binds with high affinity to P-selectinn but it can also bind E- and L-selectin. ESL-1 is a specific ligand for E-selectin. The ligands for L-selectinn on endothelial cells can be MadCAM, GlyCAM. B. p, and p2 integrins mediate firm adhesion

off leukocytes to the endothelium. Integrins are expressed by leukocytes and their ligands, from the immunoglobulinn superfamily, are expressed on endothelial cells.

LeukocyteLeukocyte adhesion to vessel wall-localized mixed thrombi: the platelet factor.factor. Remarkably, the above-described multi-step process of leukocyte adhesion

too stimulated EC is very similar to their adhesion to a thrombus formed at a damaged vascularr wall. In this respect, the thrombus seems to be a stronger adhesive substratee than the activated endothelium. In vitro experiments have identified the molecularr substrates of these interactions as specific cell adhesion molecules and ligandss present on platelets and/or leukocytes 45"49. Matrix glycoproteins in the exposedd subendothelial tissue and other specific proteins within the thrombus (e.g. fibrinn or vWF) can serve as adhesion molecules for both leukocytes and/or platelets. Platelett and fibrin/fibrinogen deposition occur at sites of vascular damage. These thrombii often become infiltrated with inflammatory cells. In vivo observations in atherosclerosis,, vasculitis, and thrombosis have shown that platelets and large numberss of leukocytes colocalise at these sites 46'50'51. Colocalisation is probably causedd by the fact that even minimal platelet deposition and activation at the injured vessell wall result in high expression of P-selectin at the platelet surface, which can servee as a strong adhesive surface for leukocytes 52. Leukocytes are able to tether overr platelet-rich thrombi and platelet aided-colocalisation of leukocytes has been shownn not only in vitro, by means of perfusion systems 52,53, but also in in-vivo grafts inn mice 54. The role of platelets and the (32-integrin Mac-1 in monocyte or PMN firm

adhesionn has been well established and involves a P-selectin-mediated stimulation off Mac-1-dependent adhesion 55. Leukocyte arrest by platelets can be mediated by

interactionss of leukocyte integrins with GPIIbllla-bound fibrinogen (Mac-1), ICAM-2 (LFA-1)) or vWF on platelets 56"61. Similar to fibrinogen, also fibrin is a very potent substratee for p2 integrin-mediated leukocyte adhesion 62. Therefore, platelets and

fibrinn are synergistic/cooperative substrates supporting leukocyte rolling/tethering interactionss and firm adhesion, respectively 63. Additionally, oriented and flow-formedd fibrin tracks have been described as very strong adhesive substrates for leukocytes. .

AtAt high shear stress, and thus bond-disturbing and detachment-inducing conditions, alll receptor-ligand interactions, in principle redundant, become more and more criticall to maintain firm adhesion. At low shear stress or static conditions, redundancy betweenn the several possible interactions allows several of these bonds to be absent beforee overall adhesion is affected.

IncreaseIncrease in adhesion due to homotypic interactions between leukocytes.

Homotypicc aggregates or cluster-forming interactions can occur at thrombi or at the endotheliall cell surface and are able to efficiently increase leukocyte adhesion 14,62,64"

67

.. Under flow conditions, adhered monocytes can serve as an L-selectin-rich surface thatt can slow down other leukocytes still in the circulation and lead to the formation off cell clusters or strings elongated according to the flow direction 67. These dusters aree L-selectin dependent and are observed mainly when the local flow is increased. Thee observed increase in adhesion is dependent on the cluster mechanism. However,, even when L-selectin function on leukocytes is blocked, they still aggregatee and interact with P-selectin glycoprotein ligand-1 (PSGL-l)-expressing cells.. Platelet microparticles (PMPs) allow neutrophils to aggregate and interact with PSGL-11 -expressing cells, indicating that leukocyte adhesion might be strengthened byy the presence of PMPs 68. PMPs are released from activated platelets and express functionall adhesion receptors, including P-selectin, on their surface. In a similar way, andd in agreement with the fact that leukocytes adhere to activated platelets on the vascularr wall, activated platelets in the circulation might also bind leukocytes and formm the so-called platelet-leukocyte complexes 13,14,69.

Platelet-leukocytee aggregates in circulation

Underr hemodynamic conditions, the rolling and arrest of leukocytes on spread plateletss or activated endothelium involves the transition from P-selectin-mediated tetheringg to more stable (32-integrin - mediated interactions. However, stimulation of

plateletss in the circulation can also occur. These platelets might then adhere to other adjacentt platelets to form platelet-platelet aggregates or to leukocytes and form

platelet-leukocytee aggregates (PLAs). PLA formation is mainly initiated via P-selectin, expressedd on platelets that will bind PSGL-1 on leukocytes 70,71 (Figure 4A). Circulatingg PLAs mostly consist of monocytes and granulocytes heterotypically aggregatedd with platelets, rather than lymphocytes 72"74. As said, platelets and leukocytess conjugate primarily via bridgings of P-selectin - PSGL-1; however, integrinmediatedd interactions via glycoprotein (GP) llb/llla fibrinogen -CD11b/CD188 75'76 and CD36 (GPIV) - thrombospondin - CD36 77 also contribute. PLAA formation seems to strongly increase upon platelet activation 72,73 and mildly uponn leukocyte activation 78"81. In fact, platelet activation is one of the major characteristicss present throughout the atherosclerotic process. Circulating activated plateletss have been found in patients with unstable atherosclerosis 82"85, stable coronaryy disease 86 and hypercholesterolemia87,88.

However,, platelet-leukocyte aggregates are not only considered as markers of platelett activation and thus of cardiovascular disease, but also as having (patho)physiologicall importance. By passive addition of platelet receptors to the aggregatee and, possibly, also by active upregulation of adhesion molecules on the leukocyte,, platelets bound to leukocytes facilitate leukocyte rolling, adhesion and migrationn on/into the vessel wall in in-vitro assays 13'1454. in agreement with this notion,, activated platelets affect endothelial inflammation and leukocyte-endothelial interactionss and, therefore, the development of atherosclerotic lesions in atherosclerosis-pronee animals 13,89. In fact, P-selectin knock-out models show a weakerr development of atherosclerosis 90. Furthermore, activated platelets also bind too circulating lymphocytes and may support lymphocyte homing to the lymph nodes

54 4

Thee in vivo circulation time and clearance of PLAs is still not well defined. Ley and co-workerss 13 have shown that circulating PLAs, in mice, were no longer detectable att 3 to 4 hours after a single injection of activated platelets. Moreover, the infusion of activatedd platelets caused removal of leukocytes, preferentially monocytes, from the circulation.. Since interactions of platelets with the vessel wall only occur in a transientt way, leading to little platelet accumulation on the endothelial surface of atheroscleroticc lesions 13'14'69, P-selectin - mediated platelet-leukocyte interactions seemm to be more stable 91"93. Probably, PLA formation will cause monocytes and neutrophilss to disappear from the circulation by adhesion of the complexes to activated,, damaged vascular wall e.g. atherosclerotic lesions in carotid arteries 13.

resting g

platelet t platelet t activation n

P-selectin n

Figuree 4. Translocation of P-selectin during platelet activation. P-selectin resides in the

alphaa granule membrane in resting platelets. Upon activation, specific proteins will form complexes andd will direct the granules to the plasma membrane. Recognition and fusion occurs and P-selectin is translocatedd to the plasma membrane where the extracellular domain can establish contact with adhesionn molecules on other cells (e.g. PSGL-1 on leukocytes). P-selectin binding to leukocyte receptorss leads to activation of proinflammatory and prothrombotic pathways by increasing the capacityy of the leukocyte to adhere secrete cytokines.

Changess in leukocyte phenotype by platelet binding and/or by paracrine platelet-derivedd factors

Too bind P-selectin, PSGL-1 has to undergo several molecular modifications. PSGL-11 is a type I transmembrane protein 94 with an extracellular domain characterizedd by repeat units that include sites for O-linked glycosylation 64<9495. Thesee are, presumably, the sites for the fucosylated, sialylated glucosamines that aree critical for P-selectin recognition 96~". Furthermore, PSGL-1 undergoes critical sulfationn of specific tyrosine residues that is required for P-selectin recognition 100'101, Besidess their docking function in cell-cell interactions, both P-selectin and PSGL-1 aree also involved in signal transduction (Figure 4B). Leukocyte activation occurs partiallyy through signaling via PSGL-1 and, therefore, platelets might be particularly efficientt because of their high surface density of P-selectin 102"105. PSGL-1 signaling iss responsible for an array of intracellular events within the leukocyte that will modify nott only the expression and affinity of adhesion molecules (e.g. integrins) but also genee transcription within monocytes, e.g. tissue factor (TF) 106-110 0

Onn the observed time scale of several hours, tissue factor upregulation might not be relevantt to local hemostasis, but after diapedesis and possibly macrophage

differentiation,, this monocyte-derived TF might become important in further thrombogenesiss and wound healing. P-selectin - mediated signaling might additionallyy prime monocytes towards pro-inflammatory responses 111. While the productionn of various cytokines is induced when leukocytes bind P-selectin through PSGL-1,, mostly widely reported effects are Mac-1 integrin activation and, with it, homotypicc neutrophil aggregation and neutrophil-platelet conjugation 75'104. Hidari et al.. 102 observed that engagement of PSGL-1 induces tyrosine phosphorylation, activatess mitogen-activated protein (MAP) kinases (ERK-1 and ERK-2) through MEK (MAPP kinase kinase), and stimulates IL-8 secretion by neutrophils. Direct correlation off these events with p2-integrin activation has been the subject of some controversy.

Somee reports concluded that PSGL-1 ligation is not sufficient to activate p2 integrins

onn neutrophils 112 while others 103 showed P-selectin induced p2 integrin-mediated celll attachment to ICAM-1. Recently, Ma et al.1 1 3 demonstrated that the binding of P-selectinn to PSGL-1 results in a moderate clustering and a partial activation of Mac-1, thuss enhancing adhesion and binding of human neutrophils to immobilized fibrinogen.. P-selectin binding also promotes VLA-4-dependent adhesion of monocytess to vascular cell adhesion molecule-1 (VCAM-1)105 under flow conditions. Anotherr interesting signaling pathway occurs via the CD40 ligand (CD40L), expressedd on the surface of activated platelets 114. Upon ligation, the cognate receptorr CD40, which is present on B cells, monocytes, macrophages and endotheliall cells, may trigger inflammatory reactions 115 by inducing expression of tissuee factor and other adhesion molecules 114'116117.

Plateletss in complex with leukocytes are likely to have paracrine effects due to the secretionn of a number of chemokines of both the CC and CXC subgroups 118, most of whichh are know to be stored in the a-granules 119_121. The chemokine RANTES is knownn to be a chemoattractant for lymphocytes, monocytes and eosinophils 119-122-124 andd to stimulate [Ca2+]j transients in these cells 125. As deposition of RANTES by plateletss triggers shear-resistant monocyte arrest on inflamed or atherosclerotic endotheliumm 126, platelet-derived RANTES in the PLAs might act similarly and supportt recruitment of monocytes from the circulation to the endothelium 127. RANTES,, in the context of PSGL-1 engagement by P-selectin, also induces MCP-1 andd IL-8 secretion from monocytes 124. Similarly, exposure of monocytes to P-selectinn and platelet activating factor (PAF) induces expression of the cytokines TNFaa and MCP-1 128.

Leukocytess may also synergistically influence platelet activation 120. Leukocytes per see and leukocyte released-02~ may enhance platelet adhesion. Furthermore,

leukocyte-releasedd substances, such as O2", platelet-activating factor (PFA), elastase,, and cathepsin G, may induce platelet aggregation and secretion 129. Conversely,, unstimulated or weakly activated leukocytes may also attenuate platelet aggregationn via leukocyte-released NO and/or ADPase 130. Neutrophil-derived

elastasee may contribute to proteolysis of the GP Iba subunit131, which contains the vonn Willebrand Factor binding site, and may thus influence platelet adhesion.

Inn conclusion, P-selectin - PSGL-1 interactions, together or not with other cooperativee factors, seem to initiate a number of signaling cascades, in both interactingg cells, that can modify or amplify a range of inflammatory and/or thrombotic responses.. The total effect of these responses will depend on the inflammatory stimulus,, the vascular bed, the inflammatory mediators presented, and the types of leukocytee recruited to the site of injury. Indeed, after the formation of PLAs, the subsequentt P-selectin - mediated leukocyte activation seems to compensate for the sheddingg of L-selectin as an important player in leukocyte tethering and rolling 14. Leukocytee activation by P-selectin binding increases the affinity/avidity of the leukocytee integrins and therefore leads to increased firm adhesion and migratory capacityy of the PLA.

{Pathophysiologicall implications

Atherosclerosis,, widely recognized as an inflammatory disease, is characterizedd by early accumulation of monocytes into the arterial wall 51 and the mechanismss that modulate this early step are thus of great importance. P-selectin wass shown to be particularly important in the pathogenesis of atherosclerosis in the apolipoproteinn E-deficient mouse. PLAs, by presenting a stronger adhesive capacity ass compared to "bare leukocytes", should be considered as proatherogenic particles thatt a. might accelerate atherothrombotic disease and b. could thus be a target for therapy.. In fact, the presence of PLAs often correlates with an adverse outcome of patientss e.g. with unstable angina pectoris. PLA formation and platelet microparticles aree also elevated in other platelet-activating conditions, and their pathophysiological rolee seems to exceed the well-established role as local atherosclerosis amplifier and triggerr of thrombosis. Increased populations of circulating, activated platelets have beenn observed in conditions such as diabetes mellitus 132, coronary restenosis 133, allergicc inflammation 134135 and severe trauma or sepsis 136. These platelets increase theirr levels of activated glycoprotein alip3, thrombospondin and most importantly,

P-selectin.. In myeloproliferative syndromes, platelet activation 137138 and PLA formation aree associated with an increased risk of arterial/venous thrombotic events 139_142. in heparin-inducedd thrombocytopenia (HIT), a strong relation between the complicationss of heparin therapy with elevated formation of PLA has been suggested

143

.. Furthermore, an increased formation of platelet-neutrophil coaggregates occurs duringg dialysis 144. This was reported to be due to increased expression of CD15s (Lewisx)) on neutrophils and monocytes, which by interacting with CD62P (P-selectin) willl play a major role in the transient leukocyte margination during hemodialysis

144,1455 |n addition a|s o patients with Alzheimer disease show elevated levels of

platelet-P-selectinn and PLAs that might result from platelet stimulation by a damaged cerebrall endothelium 146.

Pharmacologicall modulation of PLA presence or function

Thee general assumption that inflammation is important in atherothrombosis hass supported the therapeutic use of anti-inflammatory treatment in the prevention of cardiovascularr disease 147*149. indeed, there is evidence that blocking inflammation couldd lower thrombosis and thus prevent acute coronary events.

Anticoagulationn and antiplatelet therapies are known to modulate vascular death risk. However,, to what extent these therapies are effective in modulating coagulation- and platelett activation - dependent inflammation, is unknown. This should be considered whenn choosing for any classic or new therapeutic agent that blocks platelet activation and/orr coagulation, e.g. aspirin, glycoprotein GPIIbllla inhibitors or COX inhibitors. Aspirinn has become the standard antiplatelet agent for prevention of most ischemic syndromes.. Mainly, long-term therapy with aspirin reduces the risk of critical cardiovascularr and cerebrovascular events (death, stroke, myocardial infarction, unstablee angina) by average 25% compared to placebo 15a151. However, although aspirinn is a widely used platelet aggregation inhibitor, there are some studies showingg that aspirin treatment does not attenuate platelet or leukocyte activation 152, andd therefore its efficacy in preventing coronary events is being questioned. Other widelyy used platelet inhibitors are the thienopyridines (ticlopidine and clopidogrel), whichh affect platelet functionality through inhibition of ADP platelet activation. Comparedd to aspirin these agents have been shown to offer convincing and clinically significantt benefits with respect to reduction of ischemic events. More importantly, clopidogrell was shown to reduce platelet - P-selectin expression and, subsequent, platelet-leukocytee aggregate formation 153-155, decrease serum level of soluble ICAM-11 and diminish chemokinesis of monocytes 156. Furthermore, the incorporation of GP llb/lllaa receptor blockers into medical stabilization and mortality-reducing regimens forr unstable angina has enhanced the safety and has reduced the number of invasivee procedures. However, Hu and co-workers 157 showed that although GPIIb/lllaa blockade attenuated PAF-induced platelet activation and PAF-induced platelet-leukocytee aggregation, it also enhanced ADP- or TRAP-induced platelet leukocytee aggregation. Therefore, definitive conclusions about the current role of GP llb/lllaa inhibitors as inflammatory modulators are difficult to draw because many of thee studies performed to support these agents show conflicting and inconsistent results. .

Inn conclusion and although a tot of progress has been made throughout the years, theree are still controversies and conflicting research results regarding the developmentt of new therapies for management of patients at risk of vascular-related disorders.. Regarding the fact that platelets as well as leukocytes, and most importantly,, PLAs are important contributors to arterial thrombosis and atherosclerosis,, inhibition of aggregate formation might become a potent tool in the therapyy of cardiovascular diseases. Many of the therapies currently used have no effectt on the formation of PLA. In fact, only few studies have focused on the effect of therapyy on platelet interactions with leukocytes. Most of those studies only showed a decreasee in platelet - P-selectin expression and subsequent PLA formation after combiningg two or three different treatments 158"160. However, the advantages of these combinedd therapies have to be evaluated considering costs of therapy and possible toxicityy associated with hematological risks. An efficient therapy might be the use of P-selectin/PSGL-11 antagonists. It has been shown that mice deficient in P-selectin, E-selectinn or ICAM-1 are protected from a variable degree from atherosclerosis in murinee models 90,161,162. P-selectin inhibition has been shown to have advantageous resultss in enhancing thrombus resolution in rat models 163 and to decrease inflammationn and thrombus formation in baboons 164165. This effect was shown to be duee to a strong inhibition of leukocyte-endothelium interactions and subsequent reductionn in the leukocyte infiltrate into the vessel wall. This new strategy might becomee extremely useful in the treatment of cardiovascular disease in humans but clinicall studies have to be awaited. However, the optimal therapy and its overall effectivenesss are still a matter of debate.

Aimm of the study

Adhesionn of monocytes to the endothelium can be supported by monocyte-monocytee interactions, resulting in the formation of cell aggregates at the vessel wall (clusters).. Platelets that are bound to the injured vessel wall are also able to support monocytee adhesion, and platelet activation leads to the formation of platelet-monocytee complexes (PMC) in the circulation. We hypothesize that direct interactionss between platelets and monocytes enhance the monocyte atherogenic capacityy and that PMC formation might correlate with vascular disease by inducing monocytee activation and adhesion to the vessel wall.

Thiss study focuses on the differentially modulated mechanisms by which monocyte adhesion,, as primary event in atherosclerosis, can take place at the vessel wall. We havee studied monocyte interactions with the endothelium and focused on the influencee of PMC in this process. As a model, we studied monocyte/PMC rolling and adhesionn to stimulated endothelial cells under physiological flow conditions, using an in-vitroo perfusion system. In fact, flow is an important aspect of this study, because

primaryy tethering, rolling, secondary tethering and firm adhesion are steps of the multistepp model that can only be properly studied under flow conditions

Wee used a flow chamber, depicted in Figure 5, to answer the following questions; ;

What is the influence of platelet binding to monocytes on the monocyte

adhesiveadhesive capacity to the endothelium? The adhesion molecules that are involved in

platelet-monocytee complex formation and in the rolling and adhesion of monocytes/PMCC to activated endothelium were characterized (Chapter II). The presencee and new role of a specific adhesion molecule on endothelial cells were discoveredd (Chapter III).

What is the influence of platelet binding to monocytes on the monocyte

activationactivation status? The changes that occur at the monocyte surface level upon

platelett binding, regarding expression of adhesion molecules and implicating changess in the monocyte adhesive capacity, were characterized (Chapter IV).

Which other molecules or mechanisms might play an important role in

monocytemonocyte recruitment to the activated endothelium. Other important mechanisms

andd molecules, involved in leukocyte recruitment to endothelium have been described.. Molecules such as LDL-receptor - related protein (Chapter V) and LewisY (Chapterr VI) play an important role regulating/mediating leukocyte adhesion to the endothelium. .

Figuree 5. In vitro flow chamber model. Coverslips coated with a confluent layer of

endotheliall cells are placed in the flow chamber (1). Leukocytes are added to the reservoir (2) and are pulledd into, and through, the flow chamber, over the endothelial cells, by a pump (3). On the microscopee (4), a video camera (5) is mounted and with a video recorder (6) films of the rolling or adherentt cells are recorded. A heater (8) on the incubation box (9) regulates and keeps the temperaturee at .

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