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Integrin α IIb β 3 regulation in platelets Pieter E.M.H. Litjens

Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

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Page 1: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Integrin αIIbβ3 regulation in platelets

Pieter E.M.H. Litjens

Page 2: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Cover: Platelets, spreading and adhering.

Yvonne van Willigen

ISBN 90-902-1072-5

Page 3: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Integrin αIIbβ3 regulation in platelets (with a summary in English)

Integrine αIIbβ3 regulatie in trombocyten (met een samenvatting in het Nederlands)

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector

magnificus, prof. dr. W.H. Gispen, ingevolge het besluit van het college voor promoties in

het openbaar te verdedigen op vrijdag 20 oktober 2006 des ochtends te 10.30 uur

door:

Pieter Eric Marie Hubert Litjens

geboren op 5 mei 1971 te Maasbracht

Page 4: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Promotor: Prof. Dr. J.W.N. Akkerman

The results presented in this thesis were supported by the Netherlands Organisation for

Scientific Research (NWO, grant 902-26-186), the Catharijne Foundation and the Van

Walree Fund (Royal Academy of Arts and Sciences).

Financial support by Roche Diagnostics Nederland B.V., Genzyme Nederland B.V. and

Genzyme Therapeutics for the publication of this thesis is gratefully acknowledged.

Page 5: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

It's been a long road

Getting from there to here

It's been a long time

But my time is finally near

And I will see my dream come alive at last,

I will touch the sky

And they're not gonna hold me down no more

No, they're not gonna change my mind

Cause I got faith of the heart

I'm going where my heart will take me

I got faith to believe

I can do anything

I got strength of the soul

And no one's gonna bend or break me

I can reach any star

I got faith

Faith of the heart...

Diane Warren: Where My Heart Will Take Me

Page 6: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Contents

Abbreviations 7

Chapter I General introduction 8

Chapter II Integrin αIIbβ3 signalling

Part A: Integrin affinity modulation and signalling

Part B: Platelet integrin αIIbβ3: target and generator of signalling

Pieter E.M.H. Litjens, Jan-Willem N. Akkerman and Gijsbert van Willigen

Platelets, 2000, 11, 310-319

33

Chapter III Involvement of the β3 E749

ATSTFTN756

region in stabilizing the αIIbβ3-

ligand interaction

Pieter E.M.H. Litjens, Gertie Gorter, Jari Ylänne, Jan-Willem N. Akkerman and

Gijsbert van Willigen

Journal of Thrombosis and Haemostasis, 2003, 1, 2216-2224

49

Chapter IV Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in

platelet adhesion on fibrinogen under flow conditions

Pieter E.M.H. Litjens, Christine I. Kroner, Jan-Willem N. Akkerman and Gijsbert van

Willigen

Journal of Thrombosis and Haemostasis, 2003, 1, 2014-2021

59

Chapter V A tripeptide mimetic of von Willebrand factor residues 981–983

enhances platelet adhesion to fibrinogen by signalling through integrin

αIIbβ3

Pieter. E . M. H. Litjens, Gijsbert van Willigen, Cees Weeterings, Martin J . W.

IJsseldijk, Marjolein van Lier, Erkki Koivunen, Carl. G. Gahmberg and Jan-Willem N.

Akkerman

Journal of Thrombosis and Haemostasis, 2005, 3: 1274–83.

68

Chapter VI General Discussion 79

Summary 95

Samenvatting voor niet-ingewijden (Summary in Dutch) 98

Nawoord 102

Curriculum Vitae 106

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Abbreviations

aa aminoacid

AA arachidonic acid

AC adenylyle cyclase

BSA bovine serum albumine

cAMP cyclic 3’,-5’-AMP

cGMP cyclic 3’,-5’-GMP

COX cyclooxygenase

cPLA2 cytosolic phopholipase A2

DAG 1,2 sn-diacylglycerol

DMSO dimethylsulfoxide

DTS dense tubular system

ECL enhanced

chemoluminescence

ECM extra cellular matrix

EDTA ethylene-diaminetetraacetic

acid

ERK extracellular signal

regulated kinase

FACS fluorescence activated cell

sorter

FAK focal adhesion kinase

FITC fluorescein isothiocyanate

Fura-2-AM fura-2-acetoxymethyl esther

GEF guanine nucleotide exchange

factor

Gp glycoprotein

G-protein GTP-binding protein

5-HT 5-hydroxytryptamine

ICAM intercellular adhesion

molecule

ICY integrin cytoplasmic tyrosine

IP3 inositol 1,4,5,-triphosphate

MAPK mitogen activated protein

kinase

MIDAS metal-ion-dependent-

adhesion site

OCS open canicular system

PAGE polyacrylamide gel

electrophoresis

PAF platelet activating factor

PAR protease activated receptor

PBS phosphate buffered saline

PGI2, PGE2 prostaglandin I2, E1

PGG2 prostaglandin G2

PGH2 prostaglandin H2

PI-3-kinase phosphatidylinositol-3-kinase

PIP2 phosphatidylinositol 4.5-

bisphosphate

PIP3 phosphatidylinositol 3,4,5,-

trisphosphate

PKC(s) protein kinase(s) C

PKA, PKG protein kinase A, G

PLA2 phospholipase A2

PLC phospholipase C

PTB phosphotyrosine binding

PTK(s) protein tyrosine kinase(s)

PMA phorbol 12-myristate-13-

acetate

PMSF phenylmethyl-sulfonyl

fluoride

PPACK H-Phe-Pro-Arg-chlorometyl

ketone

PRP platelet rich plasma

PTP phosphotyrosine phosphatase

SH Src homology

SLO streptolysin O

SYK spleen tyrosine kinase

TxA2 thromboxane A2

VASP vasodilator stimulated

phosphoprotein

vWF von Willebrand factor

-7-

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Chapter I

General Introduction

I-1 Arterial thrombosis ........................................................................................................... 9

I-2 Haemostasis and platelet activation................................................................................ 10

I-3 Inhibition of platelet function ......................................................................................... 12

I-4 The integrin αIIbβ3 ........................................................................................................... 14

I-5 The structure of the extracellular parts of the integrin ................................................... 15

I-6 Ligand binding sites implicated in integrin recognition................................................. 17

I-7 Regulatory and divalent cation binding sites in integrins............................................... 18

I-8 Functional analysis of integrins using peptides .............................................................. 20

I-8.1 Intracellular peptides ............................................................................................... 20

I-8.2 Extracellular peptides .............................................................................................. 20

I-9 von Willebrand factor ..................................................................................................... 21

I-10 A-domains in vWF ....................................................................................................... 22

I-11 A3-domain in vWF....................................................................................................... 22

I-12 Scope of this thesis ....................................................................................................... 23

-8-

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I-1 Arterial thrombosis

Cardiovascular disease is the main cause of death in the western world. Our current lifestyle

markedly induces the risk of thrombosis and aspects like diet (high cholesterol intake), stress,

lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of

the risk factors negatively affect the vascular system, i.e. the vessel wall. Vessel wall damage

is associated with increased incidence of heart attack (myocardial infarction) or stroke, due to

the formation of arterial thrombi. Small platelet thrombi occlude the artery, which leads to

downstream ischemia. A healthy vessel wall will produce antagonists: substances that will

counteract the formation of platelet thrombi. A diseased vessel wall in this case can be

affected by two different degenerative mechanisms:

1) The vessel wall does not produce sufficient amounts of platelet pacifying antagonists.

Platelets are not kept in a quiescent state, thus small challenges lead to total activation.

In itself this does not pose a danger. However, platelets circulate with various speeds

and from ex vivo perfusion studies it has become clear that platelets behave differently

depending on the shear rates. Shear rate is defined as the relative velocity of platelets

to vessel wall. In small arteries and capillaries, a relative velocity (shear rate) of 1500-

5000 s-1

is common, in larger arteries like the aorta a shear rate of 300 s-1

is found. A

high shear rate itself puts the platelet under stress. At bifocations, where there is a

disturbance in the fluid dynamics, the shear stress is especially high. In such places,

insufficient platelet inhibition can lead to premature platelet activation, and thus

occlusion of vessels.

2) Atherosclerotic plaques have been shown to be a risk factor for arterial thrombosis.

Upon plaque rupture, the underlaying collagen fibres become exposed and lipid

metabolites become accessible to the bloodstream. The underlaying collagen and the

metabolites can both activate platelets, and certain metabolites can sensitize platelets

for activation. Atherosclerotic plaques produce several kinds of inflammatory factors.

These factors increase the prothrombotic nature of the already diseased vessel wall.

Unfortunately these two abnormalities in the vessel wall are seldom found independent from

each other. It is easy to understand that the combined effects lead to thrombotic episodes. As a

major player in arterial thrombosis, the platelet itself can be genetically prone to premature

aggregative responses. Platelets aggregate via the plasma protein fibrinogen in combination

with other plasma proteins. Responsible for this bridging is the integrin. The subunits of

integrin αIIbβ3 are polymorph in nature, with several allelic forms present in the human gene

pool. Several studies 1-4

have shown that the Pl(A2) polymorphism of the β3-subunit of αIIbβ3

is a risk factor for arterial thrombosis. Further research revealed that this polymorphism is

-9-

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responsible for an increased affinity of αIIbβ3 for fibrinogen 5. Hence this polymorphism seems

to be directly involved in increasing aggregatory responses.

In summary, differences in proximal mechanisms controlling the integrin, due to genetic

differences or differences in life style may influence the occurrence of thrombosis.

I-2 Haemostasis and platelet activation

Haemostasis is the mechanism that becomes activated upon vessel wall damage in order to

restore vessel integrity. The main players in this event are platelets. Platelets are anucleate

cells, which respond to a wide variety of agonists and adhesive proteins, after which they

aggregate and form a haemostatic plug.

Upon vessel wall injury, collagen in the subendothelium becomes exposed to the bloodstream,

which leads to the association of plasma Von Willebrand Factor (vWF) to collagen. Platelets

bind reversibly to vWF and slow down so that strong binding to collagen is possible. Platelets

change from a resting disk-shape into activated platelets, which can spread and form

pseudopods facilitating binding and aggregation. These activated platelets sequester plasma

fibrinogen and vWF, which enables them to form bridges, and thereby aggregates.

Vessel injury also initiates the coagulation cascade, resulting in the conversion of

prothrombin into thrombin. Thrombin converts fibrinogen into fibrin, which stabilizes the

thrombus. Thrombin is in addition to collagen a second platelet agonist. One of its receptors is

the protease activated receptor (PAR)-1 6. This receptor is cleaved by thrombin at Arg

41/Ser

42

and the new amino acid terminus acts as a tethered ligand, which auto-activates the PAR-1

receptor. Other receptors for α-thrombin are the GpIb-V-IX complex, PAR-37 and PAR- 4

6,8.

Platelet activation by thrombin results in the formation of thromboxane A2 (TxA2) through

activation of phospholipase C (PLC) β and γ (see below for further details). Thrombin induces

signal transduction via PLCβ, whereas collagen activates PLCγ. TxA2 is membrane

permeable so it can easily cross the platelet membrane after which the G-protein coupled

TxA2-receptor is activated. Furthermore, platelet activation results in the secretion of the

contents of three secretory vesicles inside the platelet: dense granules, α-granules and

lysosomes. Upon secretion further agonists, such as adenosine diphosphate (ADP), are

released. ADP activates platelets via two receptors belonging to the P2 purino receptor family.

Also secreted is the vasoconstrictor and platelet activator serotonin (5-hydroxy tryptamine, 5-

HT). These agonists initiate a positive feedback loop, thereby increasing platelet activation.

Platelets respond to stimulation by activating their intracellular signalling machinery. Signal

transduction transfers the signals from the outside (agonist/antagonist) into activatory

cascades and feedback loops. The concerted action of signal transduction events results in

precise, yet diverse, modulatory responses in the platelet.

-10-

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Several general ways of modulating platelet activity will be discussed below. Platelet

activation and inhibition can occur via several membrane receptors and subsequent pathways.

A schematic overview of the most essential pathways is given in figure I-1. Affinity

modulation of αIIbβ3 and signal transduction induced by αIIbβ3 will be discussed in detail in

chapter II.

Platelet activation starts with the binding of an agonist to its cognate receptor on the platelet

membrane. Most of the platelet agonists transduce signals via seven transmembrane domain

receptors that are coupled to heterotrimeric G-proteins. Several subtypes of G-proteins are

known: Gq, Gs, Gz and Gi9-12

. Heterotrimeric G-proteins contain an α-subunit that binds and

hydrolyzes GTP and a β and γ-subunit that form a dimeric complex 13

. Gi may be responsible

for the activation of PLCβ that occurs when platelets are activated by thrombin, and

simultaneously (with Gz in platelets) inhibits the formation of cAMP via adenylyl cyclase. Gq

can be activated by for instance the TxA2 receptor, so that Gαq interacts with phospholipase

Cβ, activating it and leading to Ca2+

signals as described below. The β/γ subunits of both

these G proteins may activate ras, leading to the regulation of the Mitogen Activated Protein

kinase (MAPK)-pathway.

Upon stimulation of the heterotrimeric receptor, the α-subunit exchanges GDP for GTP, and

is released in the GTP-bound state. Furthermore, the β-γ-complex is released. The liberated

subunits are second messengers and may exert either a stimulatory or inhibitory role,

depending on the receptor they were bound to. The signalling via these subunits ends when

the endogenous GTPase activity in the α-subunit hydrolyses GTP and the subunits reassociate

with the receptor.

A major effector that is activated via a heterotrimeric receptor is phospholipase Cβ. Until now

two families of PLC are identified in platelets: PLCβ and PLCγ. The PLCβ-forms are under

control of the heterotrimeric G-proteins Gq and Gi14,15

. PLCs cleave phosphatidylinositol 4,5-

bisphosphate in the membrane to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate

(IP3). DAG remains in the membrane, but IP3 enters the cytosol, binding to the IP3-receptor

on the dense tubular system. This results in the release of Ca2+

from the dense tubular system,

and subsequently an increase in the cytosolic Ca2+ 16

. The presence of both DAG and Ca2+

leads to the activation of Ca2+

-dependent isoforms of Protein Kinase C (PKC). PKC is a

Ser/Thr kinase which, when active, is capable of generating a myriad of signalling events,

ranging from granule secretion, to activation of ion-exchangers and regulation of αIIbβ3

affinity17

.

Activation by thrombin and collagen occurs also via activation of phospholipase A2 (PLA2).

PLA2 is common to many tissue types. Platelets contain two isoforms, the cytosolic PLA2

(cPLA2) and the secretable PLA2 (sPLA2). cPLA2 is activated by an increase in Ca2+ 18

, by G-

proteins 19

and by p38MAPK 17,20

. Active cPLA2 cleaves fatty acids and has a preference for

arachidonic acid (AA). After cleavage the fatty acids are mobile and for AA this mobilization

-11-

Page 12: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

results in the formation of prostaglandin G2 (PGG2) by cyclooxygenase (COX), to TxA2 via

prostaglandin H2 (PGH2) and thromboxane synthase.

cPLA2 is phosphorylated in platelets by numerous agonists. Whether there is a correlation

between phosphorylation and activity is still not clear; phosphorylation of cPLA2 occurs

independent of ERK1, p38MAPK

and PKC in platelets21

. In thrombin-stimulated platelets,

activity of cPLA2 is not inhibited by inhibition of phosphorylation.

I-3 Inhibition of platelet function

The system of haemostasis must be under tight control. Spontaneous formation of platelet

aggregates should be counteracted since this could lead to the occlusion of the vessel. This

counteraction is provided by platelet antagonists.

Like platelet agonists, some platelet antagonists inhibit platelet function by binding to a seven

transmembrane domain receptor that is coupled to a heterotrimeric G-protein. One example of

an antagonistic pathway via a seven transmembrane domain receptor is the pathway induced

by PGI2. In the case of PGI2, Gs can stimulate the formation of cAMP by adenylyl cyclase

via Gαs. Thus, G proteins can be a source of both specific direction and divergence of

signalling pathways for both activation and inhibition of platelets.

PGI2 is constitutively produced by endothelial cells in the vessel wall and is released in the

circulation. PGI2 induces a rise in platelet cAMP. A rise in cAMP is induced by prostacyclin

via the IP-receptor. This receptor is coupled to the stimulatory G-protein Gs that in the GTP-

bound form activates AC. Upon an increase of cAMP, the affinity of thrombin for its receptor

is decreased 22

, PLC activation is prevented, Ca2+

increases are inhibited, and the exposure of

ligand binding sites on αIIbβ3 is inhibited. The inhibiting effects are due to the activation of the

cAMP dependent Ser-Thr kinase protein kinase A (PKA)23

.

In addition to PGI2, endothelial cells produce nitric mono oxide (NO). NO induces a rise in

cGMP, by stimulating guanylate cyclase, which converts GTP into cGMP. This in turn leads

to activation of protein kinase G (PKG) and protein kinase A (PKA), resulting in a decrease in

intracellular Ca2+

. Both PKG and PKA have vasodilator stimulated phosphoprotein (VASP)24

as a substrate. VASP is a 50 kD protein that localizes to focal adhesions and regulates actin

and αIIbβ3 dynamics25

.

The activatory and antagonistic mechanisms are delicately balanced in haemostasis.

Premature or uncontrolled haemostatic events can lead to thrombosis, whereas inactive

haemostasis causes bleeding disorders. One such bleeding disorder originates from a defect in

αIIbβ3-fibrinogen interaction, and will be discussed below in detail.

-12-

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Figure I-1: Schematic overview of activation pathways in platelets.

Abbreviations: AA, arachidonic acid; DG, diacylglycerol; cPLA2, cytosolic phopholipase A2; IP3, inositol –1,4,5-trisphosphate; MAP, mitogen activated

protein; PGI2, prostaglandin I2; PI-3 kinase, phosphatidylinositol 3 kinase; PLC, phospholipase C; TxA2, thromboxane A2; PIP2, phosphatidylinositol-4,5-

bisphophate. Figure modified from Manning et al.11

.

-13-

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I-4 The integrin αIIbβ3

Integrins are widely expressed heterodimeric receptors, consisting of a non-covalently linked

α and β chain, that transfer signals from the outside of the cell to the inside and vice-versa.

Known combinations of α and β chains and their ligands are given in table I-1. Many β

chains associate with distinct α chains and certain α subunits associate with more than one

type of β chain and twenty-four different heterodimers have been described. While some

subfamilies are broadly expressed, e.g. β1 integrins, other subfamilies have a more restricted

tissue expression, such as β3 integrins. Many integrins bind to more than one ligand, and some

ligands bind to more than one integrin. This suggests that there is redundancy in the repertoire

of integrin-ligand interactions a cell can use for adhesive interactions in vivo, and that highly

discriminatory mechanisms determine which interaction takes place.

The functional significance of integrins has clearly been demonstrated by natural or induced

genetic defects in integrin subunits, that abolish integrin mediated adhesive functions in vivo 26

. Germ line disruptions in mice of β1 integrins are lethal 27

, demonstrating the role of β1

integrins in embryonic development. The integrin αIIbβ3 is critical in the cessation of bleeding.

Its importance is best illustrated in patients suffering from heritable Glanzmann

Thrombastenia, a qualitative or quantitative defect of αIIbβ3. Patients suffer from prolonged

and extensive bleeding and rebleeding. Timely intervention upon trauma is required since

rapid and massive blood loss can be life threatening. Several mutations resulting in different

defects have been identified. These examples of integrin defects underscore the importance of

integrins in many processes in vivo.

Activation of the integrin complex 28-31

, or inside-out signalling, comprises two ways of

action: affinity modulation, due to intensive structural change which results in stronger ligand

binding, and avidity modulation which can be described as a change in ligand-integrin contact

due to hetero- and homotypic interaction32

. The latter could be due to an increase in integrin

density at specific sites. This process is also called clustering.

The N-terminal region of αIIb (and other integrin subunits) forms a β-propeller with seven

blades oriented radially and pseudosymmetrically around an axis 33,34

. The ligand interaction

sites are located in parts that are exposed on the propeller. The β 3-I (or A) domain covers the

binding site on the propeller and thus inhibits ligand binding. Upon activation the β3-I-domain

unmasks its own intrinsic binding sites for the ligand and opens up the binding site on αIIb.

Key amino acids involved in cation-binding ligand-regulation as well as involved in ligand

recognition, are discussed below. Integrin activation and conformational changes upon that

activation and ligand binding will be discussed in detail in chapter II.

-14-

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I-5 The structure of the extracellular parts of the integrin

Early crystal structures of integrins showed a ligand binding ‘head’ and two long bent ‘legs’.

Each subunit forms one leg and the head is formed by contribution of both subunits. This

‘bent’ conformation places the ligand binding head close to the cell membrane. It is proposed

that the bent conformation is the resting state of the integrin 35,36

, and several studies have

shown that the integrin can be unbend to an extended form 37-39

. Both subunits have ‘knees’

facilitating the extension. Unmasking of the α subunits propeller is necessary for ligand

binding. In the extended position, the β-I domain lies in front of the propeller of the α subunit.

The repositioning of the β-I domain is achieved by a large outward swing of the so-called

hybrid domain 38

, which is adjacent to the I-domain in three dimensional models. This swing

induces vast structural reorganisation of the β-I domain, allowing ligands to bind with higher

affinity. The exact molecular basis of this outward swing is hitherto unknown.

It is suggested that intracellular signals separate the transmembrane domains of the two

subunits 40

. Combinations of integrin extension, outward swing and membrane separation,

give rise to the proposal of several intermediate integrin states based on the use RGD-

sequence mimetics 41

. Surprisingly, they all recognize a similar conformation. It is suggested

that the groove between the α and β subunit provides the binding site for the RGD-sequence,

and reveals a larger sub region of the α subunit that can provide a secondary ligand binding

site, the so-called cap. This model then places all binding sites on αIIbβ3 in close proximity

with each other. The RGD-sequence binding site is buried deeper in the integrin and the cap

more atop.

The secondary ligand-binding site may provide a way for the integrin to discriminate between

ligands and subsequent ligand induced signalling. Different ligands may induce different

conformations of the legs of the integrin, thereby transducing distinct signals from the binding

interface of the integrin to the inside of the cell.

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Subfamily Names Counterstructure Expression

β1 VLA-1 α1/β1

VLA-2 α2/β1

VLA-3 α3A/β1

α3B/β1

VLA-4 α4/β1

VLA-5 α5/β1

VLA-6 α6A/β1

α6B/β1

VLA-7 α7A/β1

α7B/β1

VLA-8 α8/β1

α9/β1

α10/β1

α11/β1

αV/β1

laminin, collagen

laminin, collagen

laminin, collagen, fibronectin

epiligrin, entactin

laminin, fibronectin

epiligrin, entactin

fibronectin(V25), VCAM-1, α4

fibronectin(RGD), L1

laminin

laminin

?

laminin

?

VCAM-1, osteopontin, tenascin-C

collagen

collagen

fibronectin, vitronectin

broad

broad

broad

broad

B and T lymphocytes,

macrophages, neural crest cells

broad

broad

broad

?

?

?

smooth muscle and epithelial cells,

neutrophils

broad

mesenchymally derived cells

epithelial cells

β2 LFA-1 αL/β2

CR3 αM/β2

(MAC-1)

P150,95 αX/β2

αD/β2

ICAM-1,2 & 3

C3bi, factor X, fibrinogen, ICAM-1

& 2, CD23, heparin, HMW

kininogen, NIF, denatured proteins

C3bi, fibrinogen, denatured

proteins, LPS

ICAM-3

leukocytes

granulocytes, macrophages, NK

cells, CTL

macrophages, granulocytes,

activated B lymphocytes

leukocytes

β3 gpIIb/IIa αIIb/β3

VNR αV/β3

fibrinogen, fibronectin, vW factor,

vitronectin, thrombospondin

fibrinogen, fibronectin, vW factor,

vitronectin, thrombospondin

platelets

endothelial and tumour cells,

platelets

β4 α6A/β4

α6B/β4

? (laminin)

? (laminin)

epithelial cells

epithelial cells

β5 αV/β5 vitronectin carcinoma cells

β6 αV/β6 ? ?

β7 LPAM-1 α4/β7

αE/β7

fibronectin, VCAM-1, MadCAM,

α4

E-cadherin

activated T and B lymphocytes,

macrophages, intraepithelial

lymphocytes

leukocytes

β8 αV/β8 ? ?

Table I-1: The integrin superfamily.

-16-

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I-6 Ligand binding sites implicated in integrin recognition

The main αIIbβ3 ligand is fibrinogen, a dimeric protein with each monomer consisting of an

Aα-, Bβ- and γ-chain. The dimeric structure of fibrinogen enables coupling of platelets

resulting in aggregation. Beside fibrinogen, vWF, fibronectin, thrombospondin and

vitronectin can bind to the αIIbβ3 complex. Since fibronectin, thrombospondin and vitronectin

are monomeric proteins they do not support aggregation. Ligand binding sites on αIIbβ3

consist of two types. First, RGD recognising binding sites, which recognizes the RGD

sequence in vWF, fibronectin and vitronectin42

. This sequence is also recognized by other

integrins like αIIbβ3. Second, a part of the C-terminus of fibrinogen γ-chain (γ400-411), His-His-

Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is recognized by αIIbβ3.

The sequence competes with the RGD binding site. This site is responsible for interaction of

fibrinogen and αIIbβ3 and thereby platelet aggregation 43-45

. Recently, it was shown that fibrin

binding to αIIbβ3 is mediated via a specific sequence that also becomes exposed in fibrinogen

upon immobilisation 46

. It is suggested that the encrypted sequence lies at positions 316-322

in γ-chain. Hence, studies under flow over immobilized fibrinogen provide an elegant model

for αIIbβ3 interaction with a forming thrombus, without the problem of fibrin-trapped

thrombin, as a co-stimulator. Several studies show that the RGD-sequences present in the

fibrinogen α-domain are not required for fibrinogen interaction with αIIbβ347,48

. The focus on

αIIbβ3-fibrinogen interaction is on the C-terminal part of the γ-chain49

, yet involvement of

additional sequences can not be excluded50

. Other integrins recognize different sequences on

fibrinogen such as Gly-Pro-Arg-Pro (GPRP), which is recognized by p150,50 (αxβ2) and

αMβ2.

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Figure I-2. Schematic representation of the extracellular parts of αIIbβ3 (not on scale). The αIIb subunit:

illustrated by dashed boxes are the four Ca2+

binding repeats. Point mutations discussed are indicated by arrows.

Residues involved in ligand binding are indicated with horizontal lines. The β3 subunit: the parts that form the

MIDAS-like domain are dashed. The ADMIDAS domain is found adjacent to the MIDAS sub domains. Residue

119 and 214 represent mutations found in Glanzmann Thrombastenia patients.

I-7 Regulatory and divalent cation binding sites in integrins

Generally, all integrins require divalent cations for ligand recognition and interaction. The

α subunits have seven short homologous domains of which the last three to four contain a

putative divalent binding module and are present on the lower face of the α subunit β-

propeller. Although originally thought to be EF-hand like Ca2+

-binding motifs, these sites are

now know to form a β-hairpin loops 36,51

.

The previously mentioned γ400-411

peptide, which blocks fibrinogen binding to αIIbβ3, binds to

294-314 in a Ca2+

binding repeat in αIIb (figure I-2). Peptides from this region of αIIb and

antibodies against these peptides abrogate fibrinogen binding to αIIbβ3, which emphasizes the

role of this domain in ligand binding 52

.

Several α chains contain a region of 200 amino acids that shares a structural homology to the

ligand-binding domain in vWF. This region has been designated the A or I (inserted domain).

The I domain is crucial for ligand recognition in this integrin 53

. The oxygenated residues in

the sequence Asp119

-X-Ser-X-Ser (DXSXS, X indicates a non conserved residue) plus two

non-contiguous Asp and Thr residues form within the I domain a metal-ion-dependent-

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adhesion site, or MIDAS 54

. Mutation of the MIDAS co-ordination residues leads to the loss

of ligand binding capacity in β2 integrins 55

.

Several single residues involved in ligand binding have been identified in addition to motifs

that contain cation-binding sites. Mutations in residues Gly184

, Tyr189

, Tyr190

, Phe191

and

Gly193

of αIIb abrogate binding of soluble fibrinogen to αIIbβ356

. Recently, Takada et al. 57

described an important ligand binding interface at the edge of the top and on the side of the β-

propeller by mutational analysis.

RGD-binding has been identified by chemical cross-linking to be positioned at the β3 subunit,

residues 109-171 (figure I-2). Monoclonal antibodies directed against this region inhibit

ligand binding to αIIbβ358

. Analysis of mutant αIIbβ3 with substitutions of Asp119

, Ser121

or

Ser123

has given strong evidence for a direct interaction between the ligand and this region,

since these substitutions completely abrogated ligand binding 59,60

. All these residues are part

of the DXSXS sequence of a MIDAS-like domain that is highly conserved in all integrin β

subunits. For β3 the residues that make up the MIDAS are Asp119

-X-Ser-X-Ser and in addition

Glu220

and Asp251

. The MIDAS domain is one of three cation-binding sites present on the top

face of the β-I domain. In ligands that bind via their RGD-sequence, aspartic acid of RGD co-

ordinated directly to a metal ion bound at the MIDAS, explaining the dependence of ligand

binding on divalent cations 35

. Adjacent to the MIDAS is the second site, ADMIDAS. The

ADMIDAS is formed by Ser123

, Asp126

, Asp127

, Met335

and Asp251

. Only one of the two

residues is occupied, depending whether or not ligand is bound to the integrin61

. The last is

termed LIMBS (ligand -associated metal-binding site). Three classes of cation binding have

been identified until now in α5β162

. The exact role of each cation binding class and their

molecular basis is still not fully understood.

The β-subunit I-domain of which the MIDAS-like domain is a part, has recently been

implicated in the regulation of ligand binding on the neighbouring α subunit 63

. D’Souza et

al. 64

showed elegantly with synthetic peptides corresponding to β3 residues 118-131 that both

ligand and cation binding are properties of this domain. Asp119

is a pivotal regulatory amino

acid, since mutation of this residue reduces RGD-dependent as well as RGD-independent

ligand binding. This suggests that this residue is part of a common mechanism of ligand

binding 65

.

Also, the region between Ser211

and Gly222

on the β3 subunit is involved in ligand binding.

These residues, together with the DXSXS motif, constitute a Metal Ion Dependent Adhesion

Site-like domain in the β subunit66

. Peptides homologous to region between Ser211

and Gly222

can bind to fibrinogen, and antibodies against these peptides abrogate ligand binding to αIIbβ3

67. The residues Arg

214, Asp

217, Pro

219 and Glu

220 have been identified as essential residues for

ligand binding to αIIbβ360,66,68,69

, and similar residues have been located in β2 and β5

integrins70,71

.

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I-8 Functional analysis of integrins using peptides

I-8.1 Intracellular peptides

Platelets are anucleate cells; hence mutational analysis in platelets is impossible. Several

approaches have been devised to circumvent this difficulty. Expression of mutated forms of

αIIbβ3 in cells 60,72,73

generates good assays to study the role of regions and domains, however

this is not the native environment as found in the platelet. An alternative for mutational

analysis is provided by the use of cell-permeable peptide, homologous to the regions of

interest, in cells expressing αIIbβ3 74

. Introduction of peptides homologous to domains of

interest in platelets provides an elegant way to study integrin function and ligand binding 75

.

This assay proved also helpful in studying the role the cytoplasmic tail of β3 in intracellular

signalling upon ligand binding.

I-8.2 Extracellular peptides

Besides intracellular use, peptides can be used to study regions and domains of interest on the

extracellular part of integrins as well. Previously a non-RGD-heptapeptide (LSARLAF),

designed to inhibit fibrinogen binding to αIIbβ3, has been shown to induce platelet activation

via αIIbβ376,77

. LSARLAF was designed to bind next to a presumptive fibrinogen-binding site

on the αIIb subunit. From the data discussed above it becomes clear that rather than inhibiting,

binding to this site enhances αIIbβ3 function.

A peptide homologous to GSLEVNCSTTCNQPEVGGLETSY, present in ICAM-2, a ligand

of Leukocyte Function Antigen-1 (LFA-1), activates LFA-1 by directly binding to this

integrin78-81

. This peptide binds to the I-domain of the β2-subunit and changes the affinity of

the integrin for ICAM-1, -2 and -3.

These are all examples of peptides that stimulate or enhance ligand binding. As stated above,

RGD-peptides abrogate both RGD supported as well as non-RGD supported ligand binding.

In this thesis peptides are used to clarify the regulation of ligand binding to αIIbβ3 in platelets.

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I-9 von Willebrand factor

Upon vessel wall damage collagen fibres in the subendothelium become exposed. Von

Willebrand factor (vWF; figure I-3) in plasma readily binds to collagen type I, III en VI.

Upon binding to collagen in the exposed vessel wall, vWF can bind to glycoprotein (gp) Ibα

via its A1 domain to platelets, thereby slowing down rather than arresting the platelet. When

the velocity of the platelet is sufficiently slow, interaction of glycoprotein (gp) VI and the

integrin α2β1 with collagen is possible. This results in platelet activation, which leads to the

binding of vWF and fibrinogen to integrin αIIbβ3. Function and role of integrin αIIbβ3 is

discussed previously and will be discussed in detail in chapter II.

VWF (schematic overview is given in figure I-3) is synthesized as pre-pro-vWF in

megakaryocytes 82

and endothelial cells (EC) 83,84

. The pro-peptide is cleaved by the protease

furin but the peptide remains non-covalently associated with mature vWF 85,86

. After

synthesis, approximately 95 % of vWF is constitutively secreted. The remainder is stored in

the α-granula of megakaryocytes 87

and in Weibel-Palade bodies of endothelial cells 87,88

.

The stoichiometry of the propeptide and mature vWF is in the Weibel-Palade bodies 1:1. In

vivo, the vWF plasma level is 10 µg/ml and the propeptide plasma level is 1 µg/ml. Blood

group AB persons have an average plasma level of 12.2 µg/ml, blood groups A and B 10.6

µg/ml and blood group O 8.86 µg/ml and a correlation between elevated vWF levels, and thus

blood group, and venous thrombosis has been shown 89-91

.

In mature monomeric vWF, twelve domains are have been identified (figure I-3)92

. The D3

domain contains cysteins that form the N-terminal intermolecular disulfide bridges. The D´

and the D3 domains are involved in binding FVIII 93-95

. The A1 domain binds GpIα 96,

heparin 96

, collagen type III and VI 97-99

and sulfatides 99-101

. The linker between the A1 and

A2 domains is involved in binding to leukocytes 102

. The A2 domain contains the proteolytic

cleavage site for the vWF protease 103

. The A3 domain contains the collagen binding site 97,104

. For the D4 and B domains no specific functions have been assigned. The C domains

contain the recognition sequence RGD at residues 2507-2509, a sequence used by αIIbβ3 for

binding to vWF105

.

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Figure I-3: Schematic representation (not to scale) of prepro-vWF. Indicated with scissors are the cleavage

sites. Modified from Romijn 106

(thesis).

I-10 A-domains in vWF

A-type domains (and homologous integrin I- or inserted domains) are recognized in various

ligands as well as receptors, and play an important role in ligand binding. Examples are: vWF

A1, A2 and A3 domains, integrins of the β1 family (α1β1, α 2β1, α10β1, α11β1), integrins of the

β2 family (αLβ2, αxβ2, αMβ2, αDβ2) integrin, αEβ7, but not αIIbβ3, different collagen subtypes,

complement components 107

and other proteins. A-type domains adopt a so-called Rossman or

di-nucleotide binding fold that consists of a central hydrophobic β-sheet, flanked on both

sides by amphipathic α -helices. The top face consists of loops connecting the α -helices and

the β-strands and contains the metal-ion-dependent-adhesion- site (MIDAS) motive. The

bottom face consists of loops connecting the α-helices and the β-strands and contains a

disulfide bridge.

I-11 A3-domain in vWF

The A3 domain spans residues 923 to 1109 and contains the binding site for collagen types I

and III 104,108

. The multimeric structure of vWF is important for vWF binding to collagen; the

Kd of monomeric A3 and multimeric vWF for collagen is 2 µM and 4 nM, respectively 109,110

.

Crystal structures have been solved by two groups independently 111,112

and recently113

it was

shown that two different rotamers exist for the A3 domain: one that agrees with the

orientation as found in the crystal structure and another as an exposed rotamer. The putative

vWF-A3 MIDAS motif at the top-face of A3 is formed by residues Asp934

, Ser936

, Ser938

,

Ser1005

and Thr1038

. The MIDAS motif of A3 does not contain a metal-ion. Yet the binding of

A3 to collagen type I and III is metal-ion independent 111,112

. Residues 475- 598/1018-1114 110

have been implicated as putative collagen binding sites. In another study based on

cyanogen bromide degradation residues 948-998 have been identified as a putative collagen-

binding site. Recently, 114

it was shown that an antibody directed against part of the A3-

domain inhibited thrombosis in baboons by inhibiting binding to collagen types I and III.

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I-12 Scope of this thesis

The platelet integrin αIIbβ3 mediates adhesion in vivo by binding to vWF and fibrinogen. The

aim of this study is to investigate the role of the membrane distal part of the β3 subunit of

αIIbβ3 in outside-in signalling and to determine which proteins find the basis of their action in

interaction with this region in the aftermath of ligand binding. Chapter III describes the role of

two subregions in the membrane distal part in activated platelets in supsension, investigated

by the introduction of homologous synthetic peptides via cytolytic permeabilisation. These

roles are distinctively different from each other in the timescale of platelet activation and the

signal transduction routes they are involved in. Focal adhesion kinase activation is found to be

directly involved in the early stages of ligand binding and its activation can be designated to a

region containing a trimeric motif conserved amongst β subunits. In chapter IV, the

involvement of both regions in platelets under flow bound to immobilized fibrinogen are

investigated by introduction of homologous synthestic peptides using electropermeabilisation.

The cytoskeleton plays an important role in αIIbβ3 mediated adhesion and interference with the

cytoskeleton reveals novel properties in the regulation of ligand binding of the membrane

distal part of the β3 subunit. By using a synthetic phage library to identify proteins binding to

αIIbβ3, a hitherto unknown trimeric sequence of the A3 domain in vWF was found to enhance

adhesion to fibrinogen (chapter V). Peptides containing this trimeric sequence significantly

increased platelet adhesion to fibrinogen and increased phosphorylation of a protein involved

in integrin clustering. The increase in ligand binding was only seen under static conditions

and not in suspension, and peptides alone did not activate signal transduction.

Finally, the findings from this study were compared to published data to better understand our

present insight of integrin based signalling (chapter VI).

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References

1. Weiss EJ, Bray PF, Tayback M, Schulman SP, Kickler TS, Becker LC, Weiss JL,

Gerstenblith G, Goldschmidt-Clermont PJ. A polymorphism of a platelet glycoprotein

receptor as an inherited risk factor for coronary thrombosis. N Engl J Med 1996; 334:

1090-1094.

2. Ridker PM, Hennekens CH, Schmitz C, Stampfer MJ, Lindpaintner K. PIA1/A2

polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke,

and venous thrombosis. Lancet 1997; 349: 385-388.

3. Zotz RB, Winkelmann BR, Nauck M, Giers G, Maruhn-Debowski B, Marz W, Scharf

RE. Polymorphism of platelet membrane glycoprotein IIIa: human platelet antigen 1b

(HPA-1b/PlA2) is an inherited risk factor for premature myocardial infarction in

coronary artery disease. Thromb Haemost 1998; 79: 731-735.

4. Walter DH, Schachinger V, Elsner M, Dimmeler S, Zeiher AM. Platelet glycoprotein

IIIa polymorphisms and risk of coronary stent thrombosis. Lancet 1997; 350: 1217-

1219.

5. Goodall AH, Curzen N, Panesar M, Hurd C, Knight CJ, Ouwehand WH, Fox KM.

Increased binding of fibrinogen to glycoprotein IIIa-proline33 (HPA-1b, PlA2, Zwb)

positive platelets in patients with cardiovascular disease. Eur Heart J 1999; 20: 742-

747.

6. Ramachandran R, Klufas AS, Molino M, Ahuja M, Hoxie JA, Brass LF. Release of

the thrombin receptor (PAR-1) N-terminus from the surface of human platelets

activated by thrombin. Thromb Haemost 1997; 78: 1119-1124.

7. Schmidt VA, Nierman WC, Maglott DR, Cupit LD, Moskowitz KA, Wainer JA,

Bahou WF. The human proteinase-activated receptor-3 (PAR-3) gene - Identification

within a PAR gene cluster and characterization in vascular endothelial cells and

platelets. J Biol Chem 1998; 273: 15061-15068.

8. Andrade-Gordon P, Maryanoff BE, Derian CK, Zhang HC, Addo MF, Darrow AL,

Eckardt AJ, Hoekstra WJ, McComsey DF, Oksenberg D, Reynolds EE, Santulli RJ,

Scarborough RM, Smith CE, White KB. Design, synthesis, and biological

characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. Proc

Natl Acad Sci U S A 1999; 96: 12257-12262.

9. Brass LF, Woolkalis MJ, Manning DR. Interactions in platelets between G proteins

and agonists that stimulate phospholipase C and inhibit adenylyl cyclase. J Biol Chem

1988; 263: 5348-5355.

10. Brass LF, Hoxie JA, Kieber-Emmons T, Manning DR, Poncz M, Woolkalis M.

Agonist receptors and G proteins as mediators of platelet activation. Adv Exp Med Biol

1993; 344: 17-36.

11. Brass LF, Hoxie JA, Manning DR. Signaling through G proteins and G protein-

coupled receptors during platelet activation. Thromb Haemost 1993; 70: 217-223.

-24-

Page 25: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

12. Brass LF, Manning DR, Cichowski K, Abrams CS. Signaling through G proteins in

platelets: To the integrins and beyond. Thromb Haemost 1997; 78: 581-589.

13. Akkerman JWN, Van Willigen G. Platelet activation via trimeric GTP-binding

proteins. Haemostasis 1996; 26: 199-209.

14. Smrcka AV, Sternweis PC. Regulation of purified subtypes of phosphatidylinositol-

specific phospholipase C β by G protein α and βgamma subunits. J Biol Chem 1993;

268: 9667-9674.

15. Kuang YN, Wu YQ, Smrcka A, Jiang HP, Wu DQ. Identification of a phospholipase

C β2 region that interacts with Gβgamma. Proc Natl Acad Sci USA 1996; 93: 2964-

2968.

16. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 1993; 361: 315-

325.

17. Hers I, Donath J, Van Willigen G, Akkerman JW. Differential involvement of tyrosine

and serine/threonine kinases in platelet integrin ⟨ΙΙβexposure. Arterioscler Thromb

Vasc Biol 1998; 18: 404-414.

18. Kramer RM, Sharp JD. Structure, function and regulation of Ca2+

-sensitive cytosolic

phospholipase A2 (cPLA2). FEBS Lett 1997; 410: 49-53.

19. Kim D, Lewis DL, Graziadei L, Neer EJ, Bar-Sagi D, Clapham DE. G-protein beta

gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2.

Nature 1989; 337: 557-560.

20. Börsch-Haubold AG, Kramer RM, Watson SP. Phosphorylation and activation of

cytosolic phospholipase A2 by 38-kDa mitogen-activated protein kinase in collagen-

stimulated human platelets. Eur J Biochem 1997; 245: 751-759.

21. Börsch-Haubold AG, Kramer RM, Watson SP. Cytosolic phospholipase A2 is

phosphorylated in collagen- and thrombin-stimulated human platelets independent of

protein kinase C and mitogen-activated protein kinase. J Biol Chem 1995; 270: 25885-

25892.

22. Lerea KM, Glomset JA, Krebs EG. Agents that elevate cAMP levels in platelets

decrease thrombin binding. J Biol Chem 1987; 262: 282-288.

23. Den Dekker E. Calcium and cAMP signaling during megakaryocytopoiesis. Thesis 1-

20. 2001.

24. Halbrügge M, Friedrich C, Eigenthaler M, Schanzenbächer P, Walter U.

Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets

in response to cGMP- and cAMP- elevating vasodilators. J Biol Chem 1990; 265:

3088-3093.

25. Haffner C, Jarchau T, Reinhard M, Hoppe J, Lohmann SM, Walter U. Molecular

cloning, structural analysis and functional expression of the proline-rich focal

adhesion and microfilament-associated protein VASP. EMBO J 1995; 14: 19-27.

-25-

Page 26: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

26. Fassler R, Georges-Labouesse E, Hirsch E. Genetic analyses of integrin function in

mice. Curr Opin Cell Biol 1996; 8: 641-646.

27. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by alpha 4 integrins

are essential in placental and cardiac development. Development 1995; 121: 549-560.

28. Ginsberg MH, Loftus JC, D'Souza S, Plow EF. Ligand binding to integrins: Common

and ligand specific recognition mechanisms. Cell Differ Dev 1990; 32: 203-214.

29. Ginsberg MH, O'Toole TE, Loftus JC, Plow EF. Ligand binding to integrins: Dynamic

regulation and common mechanisms. Cold Spring Harbor Symp Quant Biol 1992; 57:

221-232.

30. Ginsberg MH, Xiaoping D, O'Toole TE, Loftus JC, Plow EF. Platelet integrins.

Thromb Haemost 1993; 70: 87-93.

31. Ginsberg MH. Integrins: Dynamic regulation of ligand binding. Biochem Soc Trans

1995; 23: 439-446.

32. Hato T, Pampori N, Shattil SJ. Complementary roles for receptor clustering and

conformational change in the adhesive and signaling functions of integrin αIIbβ3. J

Cell Biol 1998; 141: 1685-1695.

33. Springer TA. Predicted and experimental structures of integrins and beta-propellers.

Curr Opin Struct Biol 2002; 12: 802-813.

34. Springer TA. Folding of the N-terminal, ligand-binding region of integrin alpha-

subunits into a beta-propeller domain. Proc Natl Acad Sci U S A 1997; 94: 65-72.

35. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, Arnaout MA.

Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with

an Arg-Gly-Asp ligand. Science 2002; 296: 151-155.

36. Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A,

Goodman SL, Arnaout MA. Crystal structure of the extracellular segment of integrin

alpha Vbeta3. Science 2001; 294: 339-345.

37. Beglova N, Blacklow SC, Takagi J, Springer TA. Cysteine-rich module structure

reveals a fulcrum for integrin rearrangement upon activation. Nat Struct Biol 2002; 9:

282-287.

38. Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in

integrin extracellular domains in outside-in and inside-out signaling. Cell 2002; 110:

599-11.

39. Chigaev A, Buranda T, Dwyer DC, Prossnitz ER, Sklar LA. FRET detection of

cellular alpha4-integrin conformational activation. Biophys J 2003; 85: 3951-3962.

40. Luo BH, Springer TA, Takagi J. A specific interface between integrin transmembrane

helices and affinity for ligand. PLoS Biol 2004; 2: e153.

-26-

Page 27: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

41. Xiao T, Takagi J, Coller BS, Wang JH, Springer TA. Structural basis for allostery in

integrins and binding to fibrinogen-mimetic therapeutics. Nature 2004; 432: 59-67.

42. Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Biol

1996; 12: 697-715.

43. Doolittle RF, Watt KW, Cottrell BA, Strong DD, Riley M. The amino acid sequence

of the alpha-chain of human fibrinogen. Nature 1979; 280: 464-468.

44. Lam SC, Plow EF, Smith MA, Andrieux A, Ryckwaert JJ, Marguerie G, Ginsberg

MH. Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain

peptides share a common binding site on platelets. J Biol Chem 1987; 262: 947-950.

45. Santoro SA, Lawing WJ, Jr. Competition for related but nonidentical binding sites on

the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins.

Cell 1987; 48: 867-873.

46. Remijn JA, Ijsseldijk MJ, van Hemel BM, Galanakis DK, Hogan KA, Lounes KC,

Lord ST, Sixma JJ, De Groot PG. Reduced platelet adhesion in flowing blood to

fibrinogen by alterations in segment gamma316-322, part of the fibrin-specific region.

Br J Haematol 2002; 117: 650-657.

47. Hantgan RR, Endenburg SC, Sixma JJ, De Groot PG. Evidence that fibrin α-chain

RGDX sequences are not required for platelet adhesion in flowing whole blood. Blood

1995; 86: 1001-1009.

48. Rooney MM, Farrell DH, van Hemel BM, De Groot PG, Lord ST. The contribution of

the three hypothesized integrin-binding sites in fibrinogen to platelet-mediated clot

retraction. Blood 1998; 92: 2374-2381.

49. Zaidi TN, McIntire LV, Farrell DH, Thiagarajan P. Adhesion of platelets to surface-

bound fibrinogen under flow. Blood 1996; 88: 2967-2972.

50. Parise LV, Steiner B, Nannizzi L, Criss AB, Phillips DR. Evidence for novel binding

sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen.

Biochem J 1993; 289: 445-451.

51. Springer TA, Jing H, Takagi J. A novel Ca2+ binding beta hairpin loop better

resembles integrin sequence motifs than the EF hand. Cell 2000; 102: 275-277.

52. Smith JW, Ruggeri ZM, Kunicki TJ, Cheresh DA. Interaction of integrins αVβ3 and

glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for

distinct binding sites. J Biol Chem 1990; 265: 12267-12271.

53. Larson RS, Corbi AL, Berman L, Springer T. Primary structure of the leukocyte

function-associated molecule-1 alpha subunit: an integrin with an embedded domain

defining a protein superfamily. J Cell Biol 1989; 108: 703-712.

54. Emsley J, King SL, Bergelson JM, Liddington RC. Crystal structure of the I domain

from integrin alpha2beta1. J Biol Chem 1997; 272: 28512-28517.

-27-

Page 28: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

55. Michishita M, Videm V, Arnaout MA. A novel divalent cation-binding site in the A

domain of the beta 2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell

1993; 72: 857-867.

56. Kamata T, Irie A, Tokuhira M, Takada Y. Critical residues of integrin alphaIIb

subunit for binding of alphaIIbbeta3 (glycoprotein IIb-IIIa) to fibrinogen and ligand-

mimetic antibodies (PAC-1, OP-G2, and LJ-CP3). J Biol Chem 1996; 271: 18610-

18615.

57. Kamata T, Tieu KK, Irie A, Springer TA, Takada Y. Amino acid residues in the alpha

IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered

in the beta-propeller model. J Biol Chem 2001; 276: 44275-44283.

58. D'Souza SE, Ginsberg MH, Burke TA, Plow EF. The ligand binding site of the platelet

integrin receptor GPIIb- IIIa is proximal to the second calcium binding domain of its αsubunit. J Biol Chem 1990; 265: 3440-3446.

59. Bajt ML, Loftus JC. Mutation of a ligand binding domain of β3 integrin. Integral role

of oxygenated residues in ⟨ΙΙβ(GPIIb-IIIa) receptor function. J Biol Chem 1994;

269: 20913-20919.

60. Baker EK, Tozer EC, Pfaff M, Shattil SJ, Loftus JC, Ginsberg MH. A genetic analysis

of integrin function: Glanzmann thrombasthenia in vitro. Proc Natl Acad Sci USA

1997; 94: 1973-1978.

61. Mould AP, Barton SJ, Askari JA, Craig SE, Humphries MJ. Role of ADMIDAS

cation-binding site in ligand recognition by integrin alpha 5 beta 1. J Biol Chem 2003;

278: 51622-51629.

62. Mould AP, Akiyama SK, Humphries MJ. Regulation of integrin alpha 5 beta 1-

fibronectin interactions by divalent cations. Evidence for distinct classes of binding

sites for Mn2+, Mg2+, and Ca2+. J Biol Chem 1995; 270: 26270-26277.

63. Yang W, Shimaoka M, Salas A, Takagi J, Springer TA. Intersubunit signal

transmission in integrins by a receptor-like interaction with a pull spring. Proc Natl

Acad Sci U S A 2004; 101: 2906-2911.

64. D'Souza SE, Haas TA, Piotrowicz RS, Byers-Ward V, McGrath DE, Soule HR,

Cierniewski C, Plow EF, Smith JW. Ligand and cation binding are dual functions of a

discrete segment of the integrin β3 subunit: Cation displacement is involved in ligand

binding. Cell 1994; 79: 659-667.

65. Takada Y, Ylanne J, Mandelman D, Puzon W, Ginsberg MH. A point mutation of

integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but

not recruitment to adhesion plaques. J Cell Biol 1992; 119: 913-921.

66. Tozer EC, Liddington RC, Sutcliffe MJ, Smeeton AH, Loftus JC. Ligand binding to

integrin ⟨ΙΙβis dependent on a MIDAS- like domain in the β3 subunit. J Biol Chem

1996; 271: 21978-21984.

-28-

Page 29: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

67. Pelletier AJ, Kunicki T, Ruggeri ZM, Quaranta V. The activation state of the integrin

⟨ΙΙβaffects outside- in signals leading to cell spreading and focal adhesion kinase

phosphorylation. J Biol Chem 1995; 270: 18133-18140.

68. Bajt ML, Ginsberg MH, Frelinger AL, III, Berndt MC, Loftus JC. A spontaneous

mutation of integrin ⟨ΙΙβ(platelet glycoprotein IIb-IIIa) helps define a ligand binding

site. J Biol Chem 1992; 267: 3789-3794.

69. Lanza F, Stierlé A, Fournier D, Morales M, André G, Nurden AT, Cazenave J-P. A

new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally

defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214

Arg-->214

Trp

mutation. J Clin Invest 1992; 89: 1995-2004.

70. Goodman TG, Bajt ML. Identifying the putative metal ion-dependent adhesion site in

the beta2 (CD18) subunit required for alphaLbeta2 and alphaMbeta2 ligand

interactions. J Biol Chem 1996; 271: 23729-23736.

71. Lin EC, Ratnikov BI, Tsai PM, Gonzalez ER, McDonald S, Pelletier AJ, Smith JW.

Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent

adhesion site-like motif but lack an I domain. J Biol Chem 1997; 272: 14236-14243.

72. Huttenlocher A, Ginsberg MH, Horwitz AF. Modulation of cell migration by integrin-

mediated cytoskeletal linkages and ligand-binding affinity. J Cell Biol 1996; 134:

1551-1562.

73. Frojmovic MM, O'Toole TE, Plow EF, Loftus JC, Ginsberg MH. Platelet glycoprotein

IIb-IIIa (⟨ΙΙβintegrin) confers fibrinogen- and activation-dependent aggregation on

heterologous cells. Blood 1991; 78: 369-376.

74. Liu XY, Timmons S, Lin YZ, Hawiger J. Identification of a functionally important

sequence in the cytoplasmic tail of integrin β3 by using cell-permeable peptide

analogs. Proc Natl Acad Sci USA 1996; 93: 11819-11824.

75. Hers I, Donath J, Litjens PE, Van Willigen G, Akkerman JW. Inhibition of platelet

integrin ⟨ΙΙβby peptides that interfere with protein kinases and the β3 tail.

Arterioscler Thromb Vasc Biol 2000; 20: 1651-1660.

76. Derrick JM, Taylor DB, Loudon RG, Gartner TK. The peptide LSARLAF causes

platelet secretion and aggregation by directly activating the integrin αIIbβ3. Biochem J

1997; 325: 309-313.

77. Derrick JM, Loudon RG, Gartner TK. Peptide LSARLAF activates αIIIbβ3 on resting

platelets and causes resting platelet aggregate formation without platelet shape change.

Thromb Res 1998; 89: 31-40.

78. Li R, Nortamo P, Valmu L, Tolvanen M, Huuskonen J, Kantor C, Gahmberg CG. A

peptide from ICAM-2 binds to the leukocyte integrin CD11a/CD18 and inhibits

endothelial cell adhesion. J Biol Chem 1993; 268: 17513-17518.

-29-

Page 30: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

79. Kotovuori A, Pessa-Morikawa T, Kotovuori P, Nortamo P, Gahmberg CG. ICAM-2

and a peptide from its binding domain are efficient activators of leukocyte adhesion

and integrin affinity. J Immunol 1999; 162: 6613-6620.

80. Li R, Xie J, Kantor C, Koistinen V, Altieri DC, Nortamo P, Gahmberg CG. A peptide

derived from the intercellular adhesion molecule-2 regulates the avidity of the

leukocyte integrins CD11b/CD18 and CD11c/CD18. J Cell Biol 1995; 129: 1143-

1153.

81. Li R, Nortamo P, Kantor C, Kovanen P, Timonen T, Gahmberg CG. A leukocyte

integrin binding peptide from intercellular adhesion molecule-2 stimulates T cell

adhesion and natural killer cell activity. J Biol Chem 1993; 268: 21474-21477.

82. Nachman R, Levine R, Jaffe EA. Synthesis of factor VIII antigen by cultured guinea

pig megakaryocytes. J Clin Invest 1977; 60: 914-921.

83. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells

derived from umbilical veins. Identification by morphologic and immunologic criteria.

J Clin Invest 1973; 52: 2745-2756.

84. Yamamoto K, de W, V, Fearns C, Loskutoff DJ. Tissue distribution and regulation of

murine von Willebrand factor gene expression in vivo. Blood 1998; 92: 2791-2801.

85. Vischer UM, Wagner DD. von Willebrand factor proteolytic processing and

multimerization precede the formation of Weibel-Palade bodies. Blood 1994; 83:

3536-3544.

86. Schlokat U, Fischer BE, Herlitschka S, Antoine G, Preininger A, Mohr G,

Himmelspach M, Kistner O, Falkner FG, Dorner F. Production of highly

homogeneous and structurally intact recombinant von Willebrand factor multimers by

furin-mediated propeptide removal in vitro. Biotechnol Appl Biochem 1996; 24 ( Pt 3):

257-267.

87. Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand protein

in Weibel-Palade bodies of human endothelial cells. J Cell Biol 1982; 95: 355-360.

88. Wagner DD. Cell biology of von Willebrand factor. Annu Rev Cell Biol 1990; 6: 217-

246.

89. Morelli VM, De Visser MC, Vos HL, Bertina RM, Rosendaal FR. ABO blood group

genotypes and the risk of venous thrombosis: effect of factor V Leiden. J Thromb

Haemost 2005; 3: 183-185.

90. Schleef M, Strobel E, Dick A, Frank J, Schramm W, Spannagl M. Relationship

between ABO and Secretor genotype with plasma levels of factor VIII and von

Willebrand factor in thrombosis patients and control individuals. Br J Haematol 2005;

128: 100-107.

91. Souto JC, Almasy L, Soria JM, Buil A, Stone W, Lathrop M, Blangero J, Fontcuberta

J. Genome-wide linkage analysis of von Willebrand factor plasma levels: results from

the GAIT project. Thromb Haemost 2003; 89: 468-474.

-30-

Page 31: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

92. Verweij CL, Diergaarde PJ, Hart M, Pannekoek H. Full-length von Willebrand factor

(vWF) cDNA encodes a highly repetitive protein considerably larger than the mature

vWF subunit. EMBO J 1986; 5: 1839-1847.

93. Kaufman RJ, Dorner AJ, Fass DN. von Willebrand factor elevates plasma factor VIII

without induction of factor VIII messenger RNA in the liver. Blood 1999; 93: 193-

197.

94. Foster PA, Fulcher CA, Marti T, Titani K, Zimmerman TS. A major factor VIII

binding domain resides within the amino-terminal 272 amino acid residues of von

Willebrand factor. J Biol Chem 1987; 262: 8443-8446.

95. Takahashi Y, Kalafatis M, Girma JP, Sewerin K, Andersson LO, Meyer D.

Localization of a factor VIII binding domain on a 34 kilodalton fragment of the N-

terminal portion of von Willebrand factor. Blood 1987; 70: 1679-1682.

96. Sixma JJ, Schiphorst ME, Verweij CL, Pannekoek H. Effect of deletion of the A1

domain of von Willebrand factor on its binding to heparin, collagen and platelets in

the presence of ristocetin. Eur J Biochem 1991; 196: 369-375.

97. Roth GJ, Titani K, Hoyer LW, Hickey MJ. Localization of binding sites within human

von Willebrand factor for monomeric type III collagen. Biochemistry 1986; 25: 8357-

8361.

98. Hoylaerts MF, Yamamoto H, Nuyts K, Vreys I, Deckmyn H, Vermylen J. von

Willebrand factor binds to native collagen VI primarily via its A1 domain. Biochem J

1997; 324 ( Pt 1): 185-191.

99. Fujimura Y, Titani K, Holland LZ, Roberts JR, Kostel P, Ruggeri ZM, Zimmerman

TS. A heparin-binding domain of human von Willebrand factor. Characterization and

localization to a tryptic fragment extending from amino acid residue Val-449 to Lys-

728. J Biol Chem 1987; 262: 1734-1739.

100. Christophe O, Obert B, Meyer D, Girma JP. The binding domain of von Willebrand

factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and

collagen and resides between amino acid residues Leu 512 and Lys 673. Blood 1991;

78: 2310-2317.

101. Data RE, Williams SB, Roberts DD, Gralnick HR. Platelets adhere to sulfatides by

von Willebrand factor dependent and independent mechanisms. Thromb Haemost

1991; 65: 581-587.

102. Koivunen E, Ranta TM, Annila A, Taube S, Uppala A, Jokinen M, Van Willigen G,

Ihanus E, Gahmberg CG. Inhibition of beta(2) integrin-mediated leukocyte cell

adhesion by leucine-leucine-glycine motif-containing peptides. J Cell Biol 2001; 153:

905-916.

103. Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE, Fujikawa K. Structure of

von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in

thrombotic thrombocytopenic purpura. J Biol Chem 2001; 276: 41059-41063.

-31-

Page 32: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

104. Lankhof H, van Hoeij M, Schiphorst ME, Bracke M, Wu YP, Ijsseldijk MJ, Vink T,

De Groot PG, Sixma JJ. A3 domain is essential for interaction of von Willebrand

factor with collagen type III. Thromb Haemost 1996; 75: 950-958.

105. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol

Chem 2000; 275: 21785-21788.

106. Romijn R. Structural studies on the von Willebrand Factor A1 and A3 domains. 2002.

107. Colombatti A, Bonaldo P, Doliana R. Type A modules: interacting domains found in

several non-fibrillar collagens and in other extracellular matrix proteins. Matrix 1993;

13: 297-306.

108. Cruz MA, Yuan H, Lee JR, Wise RJ, Handin RI. Interaction of the von Willebrand

factor (vWF) with collagen. Localization of the primary collagen-binding site by

analysis of recombinant vWF A domain polypeptides. J Biol Chem 1995; 270: 10822-

10827.

109. van der Plas RM, Gomes L, Marquart JA, Vink T, Meijers JC, De Groot PG, Sixma

JJ, Huizinga EG. Binding of von Willebrand factor to collagen type III: role of

specific amino acids in the collagen binding domain of vWF and effects of

neighboring domains. Thromb Haemost 2000; 84: 1005-1011.

110. Cruz MA, Yuan H, Lee JR, Wise RJ, Handin RI. Interaction of the von Willebrand

factor (vWF) with collagen. Localization of the primary collagen-binding site by

analysis of recombinant vWF A domain polypeptides. J Biol Chem 1995; 270: 19668.

111. Huizinga EG, Martijn vdP, Kroon J, Sixma JJ, Gros P. Crystal structure of the A3

domain of human von Willebrand factor: implications for collagen binding. Structure

1997; 5: 1147-1156.

112. Bienkowska J, Cruz M, Atiemo A, Handin R, Liddington R. The von willebrand factor

A3 domain does not contain a metal ion-dependent adhesion site motif. J Biol Chem

1997; 272: 25162-25167.

113. Hellings M, Engelborghs Y, Deckmyn H, Vanhoorelbeke K, Schiphorst ME,

Akkerman JW, De Maeyer M. Experimental indication for the existence of multiple

Trp rotamers in von Willebrand Factor A3 domain. Proteins 2004; 57: 596-601.

114. Vanhoorelbeke K, Depraetere H, Romijn RA, Huizinga EG, De Maeyer M, Deckmyn

H. A consensus tetrapeptide selected by phage display adopts the conformation of a

dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the

von Willebrand factor-collagen interaction. J Biol Chem 2003; 278: 37815-37821.

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Chapter II

Integrin αIIbβ3 signalling

Part A: Integrin affinity modulation and signalling

Part B: Platelet integrin αIIbβ3: target and generator of signalling

Pieter E.M.H. Litjens, Jan-Willem N. Akkerman and Gijsbert van Willigen

Platelets, 2000, 11, 310-319

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This chapter contains a short summary of recent insights in integrin affinity modulation,

followed by an overview of signal transduction towards αIIbβ3 and originating from αIIbβ3,

inside out and outside in signalling, respectively.

Integrin affinity modulation

In addition to expression of integrins, many cells have the ability of rapidly changing the

affinity of a given integrin for ligands as a discriminatory mechanism for adhesive behaviour.

Such affinity modulation has been described for β1 integrin 1, β2 integrins

2,3 and αIIbβ3

4,5.

Upon activation of the platelet, αIIbβ3 is changed from a low affinity to a high affinity state,

which enables the integrin to bind its ligands 6-9

. This is achieved by rapid, reversible changes

in the conformation of extracellular domains of the integrin. Generally this is called integrin

activation10,11

. Activation was used in early studies of αIIbβ312,13

to describe changes necessary

for ligand binding. Currently, the term integrin activation and the molecular basis of integrin

activation are under much debate. In the past integrin activation was described in a ‘hinge

model’ 14

, in which the cytoplasmic parts act like scissors. When the handles are closed, the

extracellular part of the integrin is opened up for ligand binding. Recently, a different ‘jack-

knife’ model 15

is favoured, where the two integrin subunits flip upwards upon activation,

hereby exposing their ligand-binding sites. The jack-knife is initiated by a downward pull of

the β chain: the piston model 16

. For this the interaction between the integrin α and β subunits

has to be disrupted, as is illustrated by the fact that mutations in and deletion of the

intramembrane parts that activate the integrin block this process 17-20

.

Currently, the focus on integrin affinity modulation is directed towards talin, rather than

previously described activation models. Due to the consensus sequence found in integrins

regarding the talin-binding site it is suggested that talin is a common mechanism in integrin

activation. The pull down of the β subunit, i.e. the piston model, is postulated to be generated

by talin, which binds to the cytoplasmic tail of the β subunit 11

. Talin is a major cytoskeletal

actin-binding protein that binds to integrin and co-localizes with activated integrins 21-26

. The

antiparallel homodimer talin consists of two subunits, with each a N-terminal globular head

(47 kD) and a 190 kD C-terminal rod domain. Talin binds to a variety of integrins

(β1A/D, β2 and β3 27

) and this binding is mediated via (Band) 4.1, Ezrin, Radixin and Moesin

homology (FERM) domain in the talin head. The C-terminal rod domain interacts with the

cytoskeleton. The subunits of the talin head contain phosphotyrosine-binding domains (PTB)

for the binding of the phosphorylated tyrosines that are found on the β3-tail upon integrin

activation. When the talin head is over expressed, integrins become constitutively active,

indicating that the talin binding site is encrypted in the resting integrin 28

. Strikingly, all

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mutations in the β3-cytoplasmic tail that abolish talin binding also abrogate integrin activity,

whereas mutations in the non-talin-binding area do not affect integrin activation.

Talin binding to the β tail generates signals that affect the membrane proximal regions29,30

.

This change in the membrane proximal parts of the β subunit leads to the separation of the α

and β subunit, generally accepted as a key step in integrin activation.

The talin head domain has a six fold higher affinity for the β3 subunit than intact talin. This

suggests that the integrin binding site on intact talin is masked 28

. The protease calpain has

been implicated to be involved in integrin activation 31-33

, and it has been shown that calpain

cleavage of talin increases talin binding to integrin in vitro28

. This suggests that in vivo

calpain might cleave talin, which then activates the integrin. Another way of unmasking the

integrin-binding site on talin is by binding of PtdIns(4,5)P2. This binding unmasks the binding

site in the FERM domain, and it has been shown that upon PtdIns(4,5)P2 binding to talin,

association of talin to β1 tails is possible 34

.

Figure 2-1: Talin mediated integrin activation. Talin is activated either by calpain cleavage or PtdIns(4,5)P2

followed by binding of talin to the β subunit of the integrin. Talin binding results in the ‘piston action’, exposing

the ligand binding sites on the extracellular parts of the integrin (jack knife model). All three steps are delineated

in the model above (modified from Calderwood 35

).

The increasing emphasis on talin as a common denominator in integrin activation implies that

activation of talin and signalling events upon integrin affinity modulation require integrin

specific effectors. Signalling events resulting in affinity modulation of αIIbβ3 and subsequent

outside-in signalling will be discussed next.

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References

1. Faull RJ, Kovach NL, Harlan JM, Ginsberg MH. Affinity modulation of integrin alpha 5

beta 1: regulation of the functional response by soluble fibronectin. J Cell Biol 1993;

121: 155-162.

2. Altieri DC, Morrissey JH, Edgington TS. Adhesive receptor Mac-1 coordinates the

activation of factor X on stimulated cells of monocytic and myeloid differentiation: an

alternative initiation of the coagulation protease cascade. Proc Natl Acad Sci U S A

1988; 85: 7462-7466.

3. Lollo BA, Chan KW, Hanson EM, Moy VT, Brian AA. Direct evidence for two affinity

states for lymphocyte function-associated antigen 1 on activated T cells. J Biol Chem

1993; 268: 21693-21700.

4. Phillips DR, Charo IF, Scarborough RM. GPIIb-IIIa: the responsive integrin. Cell 1991;

65: 359-362.

5. Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and

epinephrine. J Clin Invest 1979; 64: 1393-1401.

6. Humphries MJ. The molecular basis and specificity of integrin-ligand interactions. J

Cell Sci 1990; 97: 585-592.

7. Humphries MJ. Mechanisms of ligand binding by integrins. Biochem Soc Trans 1994;

22: 275-282.

8. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;

69: 11-25.

9. Phillips DR, Charo IF, Scarborough RM. GPIIb-IIIa: The responsive integrin. Cell

1991; 65: 359-362.

10. Sims PJ, Ginsberg MH, Plow EF, Shattil SJ. Effect of platelet activation on the

conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem

1991; 266: 7345-7352.

11. Woodside DG, Liu S, Ginsberg MH. Integrin activation. Thromb Haemost 2001; 86:

316-323.

12. Coller BS. A new murine monoclonal antibody reports an activation-dependent change

in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa

complex. J Clin Invest 1985; 76: 101-108.

13. Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane

glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-

11114.

14. Loftus JC, Liddington RC. Cell adhesion in vascular biology. New insights into

integrin-ligand interaction. J Clin Invest 1997; 99: 2302-2306.

-36-

Page 37: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

15. Humphries MJ, McEwan PA, Barton SJ, Buckley PA, Bella J, Mould AP. Integrin

structure: heady advances in ligand binding, but activation still makes the knees wobble.

Trends Biochem Sci 2003; 28: 313-320.

16. Woodside DG, Liu S, Ginsberg MH. Integrin activation. Thromb Haemost 2001; 86:

316-323.

17. Ylanne J, Chen Y, O'Toole TE, Loftus JC, Takada Y, Ginsberg MH. Distinct functions

of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation

of focal adhesions. J Cell Biol 1993; 122: 223-233.

18. Lu C, Takagi J, Springer TA. Association of the membrane proximal regions of the

alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state. J

Biol Chem 2001; 276: 14642-14648.

19. O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, Quaranta V, Loftus JC, Shattil SJ,

Ginsberg MH. Integrin cytoplasmic domains mediate inside-out signal transduction. J

Cell Biol 1994; 124: 1047-1059.

20. O'Toole TE, Mandelman D, Forsyth J, Shattil SJ, Plow EF, Ginsberg MH. Modulation

of the affinity of integrin alpha IIb beta 3 (GPIIb-IIIa) by the cytoplasmic domain of

alpha IIb. Science 1991; 254: 845-847.

21. Lankhof H, van Hoeij M, Schiphorst ME, Bracke M, Wu YP, Ijsseldijk MJ, Vink T, De

Groot PG, Sixma JJ. A3 domain is essential for interaction of von Willebrand factor

with collagen type III. Thromb Haemost 1996; 75: 950-958.

22. Calderwood DA, Ginsberg MH. Talin forges the links between integrins and actin. Nat

Cell Biol 2003; 5: 694-697.

23. Nayal A, Webb DJ, Horwitz AF. Talin: an emerging focal point of adhesion dynamics.

Curr Opin Cell Biol 2004; 16: 94-98.

24. Tremuth L, Kreis S, Melchior C, Hoebeke J, Ronde P, Plancon S, Takeda K, Kieffer N.

A fluorescence cell biology approach to map the second integrin-binding site of talin to

a 130-amino acid sequence within the rod domain. J Biol Chem 2004; 279: 22258-

22266.

25. Smith A, Carrasco YR, Stanley P, Kieffer N, Batista FD, Hogg N. A talin-dependent

LFA-1 focal zone is formed by rapidly migrating T lymphocytes. J Cell Biol 2005; 170:

141-151.

26. Ratnikov BI, Partridge AW, Ginsberg MH. Integrin activation by talin. J Thromb

Haemost 2005; 3: 1783-1790.

27. Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J

Cell Sci 2000; 113 ( Pt 20): 3563-3571.

28. Yan B, Calderwood DA, Yaspan B, Ginsberg MH. Calpain cleavage promotes talin

binding to the beta 3 integrin cytoplasmic domain. J Biol Chem 2001; 276: 28164-

28170.

-37-

Page 38: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

29. Vinogradova O, Velyvis A, Velyviene A, Hu B, Haas T, Plow E, Qin J. A structural

mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its

cytoplasmic face. Cell 2002; 110: 587-597.

30. Ulmer TS, Calderwood DA, Ginsberg MH, Campbell ID. Domain-specific interactions

of talin with the membrane-proximal region of the integrin beta3 subunit. Biochemistry

2003; 42: 8307-8312.

31. Inomata M, Hayashi M, Ohno-Iwashita Y, Tsubuki S, Saido TC, Kawashima S.

Involvement of calpain in integrin-mediated signal transduction. Arch Biochem Biophys

1996; 328: 129-134.

32. Montsarrat N, Racaud-Sultan C, Mauco G, Plantavid M, Payrastre B, Breton-Douillon

M, Chap H. Calpains are involved in phosphatidylinositol 3',4'-bisphosphate synthesis

dependent on the αIIbβ3 integrin engagement in thrombin-stimulated platelets. FEBS Lett

1997; 404: 23-26.

33. Schoenwaelder SM, Yuan Y, Jackson SP. Calpain regulation of integrin alpha IIb beta 3

signaling in human platelets. Platelets 2000; 11: 189-198.

34. Martel V, Racaud-Sultan C, Dupe S, Marie C, Paulhe F, Galmiche A, Block MR,

Albiges-Rizo C. Conformation, localization, and integrin binding of talin depend on its

interaction with phosphoinositides. J Biol Chem 2001; 276: 21217-21227.

35. Calderwood DA. Integrin activation. J Cell Sci 2004; 117: 657-666.

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Pieter E.H.M. Litjens, Jan-Willem N. Akkerman, Department ofHaematology, Laboratory for Thrombosis and Haemostasis, UniversityMedical Center Utrecht, Utrecht, The Netherlands and Institute forBiomembranes, University of Utrecht, Utrecht, The Netherlands;

Gijsbert van Willigen, Department of Haematology, Laboratory forThrombosis and Haemostasis, University Medical Center Utrecht,Utrecht, The Netherlands, Institute for Biomembranes, University of

Utrecht, Utrecht, The Netherlands and Department of Biosciences,Division of Biochemistry, University of Helsinki, Helsinki, Finland.

Correspondence to: G. van Willigen, Department of Haematology

HP G03.647, Laboratory for Thrombosis and Haemostasis, UniversityMedical Center Utrecht, P.O. Box 85.500, NL–3508 GA Utrecht, TheNetherlands. Tel: +31 30 2507610; Fax: +31 30 2511893; E-mail:

[email protected]

Review

Platelet integrin aIIbb3 : target and generator ofsignalling

Pieter E.H.M. Litjens, Jan-Willem N. Akkerman, Gijsbert van Willigen

Introduction

The integrin aIIbb3 , also known as glycoprotein (GP)

IIb/IIIa or CD41/CD61, is the most abundant membrane

protein complex on platelets. On the surface of the

platelet about 50,000 copies are present and in the open

canalicular system and a-granules another pool of about

50,000 copies is located. This platelet-specific integrin

plays an essential role in haemostasis and thrombosis as

it mediates platelet aggregation and platelet spreading on

different proteins present in the vessel wall.

Integrin aIIbb3 is also the centre of many signalling

events. Signals from the inside of the platelet are directed

to the integrin (inside-out signalling) and induce an

affinity change in aIIbb3 , enabling ligand binding. The

integrin can also be the source of signals following

ligand binding and clustering (outside-in signalling)

which results in an avidity change of the integrin and

aggregation and spreading of platelets.

Signals with aIIbb3 as a target

On a resting platelet, the aIIbb3 complex is in a non-

activated state and unable to bind ligands, such as

fibrinogen and von Willebrand factor (vWF), in a soluble

form. However, in this state the integrin is capable of

binding when these ligands are coated to a surface. After

activation of the cells by agonists such as a-thrombin

and ADP, aIIbb3 changes its conformation1 and is shifted

to a high affinity state, making binding of soluble ligands

possible. Also high shear forces can increase the affinity

state of the integrin for soluble ligands resulting in

platelet aggregation.2

Although aIIbb3 is one of the best studied integrins,

the signalling routes and the target domains of these

routes on the integrin complex that induce the affinity

change are still poorly understood. One of the signalling

pathways involved in affinity change involves protein

kinase C (PKC). This is probably the main signalling

route for agonists like a-thrombin. Activation of PAR1

and PAR4, the thrombin receptors on human platelets,3,4

results in activation of a heterotrimeric G-protein. Most

likely this is the Gq-protein since knock-out mice that

lack this G-protein do not aggregate in response to

a-thrombin.5 Activation of Gq leads to activation phos-

pholipase Cb2 (PLCb2 ) and PKC. There is evidence that

PKC directly phosphorylates the b3-chain.6,7 Residue

Thr753 is phosphorylated upon a-thrombin stimulation8

and the stoichiometry of this phosphorylation is 0.8,6

indicating that the majority of b3-chains are phosphory-

lated. Furthermore, inhibition of de-phosphorylation of

b3 made the reversible exposure seen after stimulation

by platelet-activating factor (PAF) irreversible6 as also

seen after platelet stimulation with a-thrombin.6,9

Other evidence for the involvement of PKC comes

from studies with activators and inhibitors of the

enzyme. Phorbol esters induce aggregation and ligand

binding6,10,11 and specific inhibitors of PKC block

a-thrombin-induced fibrinogen binding and phosphor-

ylation of b3 in parallel.6,12 Also a PKC-inhibiting

peptide introduced in Streptolysin O (SLO) perme-

abilised platelets inhibited a-thrombin-induced ligand

binding.13

It is unlikely that the route via heterotrimeric

G-proteins and PKC is the single pathway to an affinity

change of aIIbb3 . When agonists such as ADP were used

inhibitors of Tyr-kinases blocked the activation of aIIbb3 ,

while PKC inhibitors had no effect.10 Surprisingly, these

ISSN 0953-7104 print/ISSN 1369-1635 online/00/060310-10 © 2000 Taylor & Francis Ltd

Platelets (2000) 11, 310–319

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inhibitors also blocked a-thrombin-induced integrin

activation.3,10 Similar results were obtained with an

artificial substrate peptide for the Src-family of kinases

in SLO permeabilised platelets.13 This led to the

conclusion that Tyr-kinases, probably of the Src-family

of kinases, mediate the primary signals to activate of

aIIbb3 , whereas PKC is required for the stabilisation of

the ligand binding (Fig. 1).

Also collagen induces ligand binding to aIIbb3

although the collagen receptors on platelets, GP VI and

integrin a2b1 , do not signal via trimeric G-proteins, but

do so via Tyr-kinases. Stimulation of platelets by

collagen activates the Tyr-kinase-dependent PLC-sub-

type PLCg2 .14 –16

There does not seem to be a target for Tyr-kinases on

the b3-cytoplasmic tail that might change integrin

affinity when b3 is in complex with aIIb .7,17 In the initial

stages of activation only Thr-phosphorylation can be

observed and there is no Tyr-phosphorylation .8 At later

stages Tyr-residues become phosphorylated,18 which

appears unrelated to affinity changes, but essential for

integrin clustering, resulting in an avidity change and

signal generation by the integrin (see below).

There is also evidence that other cytoplasmic proteins

can be the target of signalling routes which upon

activation bind to one of the cytoplasmic tails (Fig. 2).

Calcium and Integrin Binding protein (CIB) binds

specifically to the aIIb-subunit19 and b3-endonexin binds

to b3 .20 Overexpression of b3-endonexin in aIIbb3

expressing CHO cells resulted in a higher affinity state of

the integrin and the subsequent binding of fibrinogen to

the cells.21

Recently, a new mechanism of aIIbb3 activation was

proposed by Bennett and co-workers.22 They showed

that one of the mechanisms by which the integrin

complex is kept in a low affinity state is attachment to

cytoskeletal proteins such as talin (probably via the

talin head) a-actinin, filamin and skelemin.23 – 27

Release from the cytoskeleton induced fibrinogen bind-

ing. This indicates that an increase in avidity may be

the cause rather than a consequence of ligand binding.

When the complex is coupled to the cytoskeleton it is

unable to move freely through the plain of the mem-

brane.28 Release from the cytoskeleton increases the

lateral diffusion of the integrin,28 which makes it easier

for integrins to cluster. At present, it is difficult to

understand how the signalling pathways initiate this

release. Probably a primary change in conformation in

the cytoplasmic tails of aIIbb3 loosens the complex

from the cytoskeleton, thereby inducing a change in

avidity that facilitates the ligand binding. A phosphor-

ylation reaction might be the primary trigger, because

all agonists we have tested (a-thrombin, ADP and PAF)

induced b3-phosphorylation.6,7 Whether the change in

avidity is really important for the initial stages of

ligand binding to aIIbb3 is doubtful. The group of

Shattil showed that the artificial clustering of aIIbb3

could not account for the number of fibrinogen binding

sites that are exposed after agonists’ stimulation.29 This

led to a model in which the affinity change of aIIbb3

was the primary regulator of ligand binding and the

change in avidity the trigger for irreversible ligand

binding.

It is not so simple to identify the target domains of the

signalling routes that trigger the affinity change. Because

aIIb has no potential phosphorylation sites most of the

studies have been focussed on b3 , which contains 1 Ser-,

7 Thr- and 2 Tyr-residues in its cytoplasmic tail (Fig. 2).

Using mutational analysis in aIIbb3-transformed CHO

cells the last six amino acids (aa) of the b3-cytoplasmic

tail were found to be essential in aIIbb3-mediated

adhesion to fibrinogen.30 Shorter truncations were less

effective or ineffective.30 Also the region around Ser752

appears important. Platelets of a Glanzmann’s Throm-

bastenia patient, an inherited bleeding disorder, with the

Ser752®Pro substitution in the b3-cytoplasmic tail do not

respond to stimulation with aggregation.31 In CHO cells,

following adhesion to fibrinogen, this mutation com-

pletely inhibited cell spreading.32,33 This does not

indicate that Ser752 has a critical importance for the

PLATELETS 311

Figure 1. Model depicting the regulation of aIIbb3 by Tyr-kinases and protein kinase C. Following platelet activation Src-family kinasesand protein kinase C (PKC) become activated. Src kinase activation (1) appears a general property of platelet agonists while PKCactivation (2) differs among various agonists. Activation of PKC by phorbol esters (3) can replace step (1). Tyr-phosphorylated integrinregulatory proteins change the conformation from a closed (aIIbb3

0 ) to an exposed one (aIIbb3*), which in the absence of a ligand rapidlyclose, owing to tyrosine phosphatase (TP) activity. Depending on the degree of PKC activation, the internal pool of aIIbb3 becomesexposed to the surface (4) and the b3-cytoplasmic tail becomes phosphorylated on Thr753 (5). This phosphorylation opposes the returnof the exposed aIIbb3 to the closed conformation. Dephosphorylation by Ser/Thr-phosphatases, probably the PP1 subtype, converts theintegrin to a closed conformation.

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affinity change of the integrin as substitution by an Ala-

residue had no effect.30,34,35 The Ser752®Pro mutation

results in a completely different conformation of the b3

cytoplasmic tail whereas the Ser752®Ala substitution

has only minimal consequences. The Pro-residue induces

the start a new b-turn in the b3-cytoplasmic tail, which

has a massive impact on the conformation. These results

point to a critical conformational constraint the b3-cyto-

plasmic tail should be in before the integrin complex can

be activated. This also indicates that results from aa

substitution studies should be interpreted with care.

Another approach to identify domains of the b3-cyto-

plasmic tail important for affinity regulation is the

introduction of peptides that mimic parts of the b3-tail.

Three research groups using different approaches came to

similar conclusions. The group of Hawiger made cell-

permeable peptides by combining the hydrophobic region

of the signal peptide of b3 with sequences of the

cytoplasmic region of b3 .36 These peptides were then

introduced in HEL-cells and their activity was analysed in

a cell adhesion assay. They showed that a peptide

containing the Thr753 (Y747KEATSTFTNITYRGT762 )

inhibited adhesion to fibrinogen, whereas the Ser752 was

not essential for integrin activation. Zhang and co-

workers linked peptides that mimicked parts of the

b3-cytoplasmic tail with the signal peptide of b3 using a

non-peptide thiazolidino linkage.37 The results were

similar showing that the region Y747KEATSTFTNI-

TYRGT762 is important for integrin activation, though

Ser752 was not essential. Our own group used SLO

permeabilised platelets to introduce b3-mimicking pep-

tides into the cytosol.13 Two peptides mimicking aa

stretches of the b3-cytoplasmic tale, E749ATSTFTN756

and T755NITYRGT762, completely blocked a-thrombin-

induced ligand binding to aIIbb3 . The peptides did not

interfere with the signal generation by thrombin-recep-

tors, PKC activation and the activation of Tyr-kinases.

The peptide containing the Thr753 appeared to be more

potent than the peptide mimicking the last eight aa of b3 .

It is not entirely clear how the peptides interfere with

the affinity change of aIIbb3 . One hypothesis is that the

peptides act as artificial substrates for the kinases that

phosphorylate b3 , explaining why the peptide with

Thr753 shows the strongest inhibition. Alternatively, the

peptides may act as fake binding sites for b3 binding

proteins such as b3-endonexin. The peptide T755NI-

TYRGT762 contains part of the binding site for b3-endo-

nexin, the protein that can increase integrin affinity.21,38

However, b3-endonexin binding to b3 and ligand binding

induced by b3-endonexin were markedly reduced by the

Ser752®Pro substitution, suggesting that the binding of

b3-endonexin also depends on the conformation of the

b3-cytoplasmic tail.21,38

Another elegant approach has been used by Baker et

al.39 These workers applied a random mutagenesis

approach on a mutationally activated form of aIIbb3 in

CHO cells. The cells that lost the ability to bind

fibrinogen were further characterised. Besides truncation

mutants she found that the point mutation Pro745®Ser

interfered with integrin activation. Probably, this muta-

tion changes the conformation of the cytoplasmic tail. A

surprising observation was that also mutations in a 23-aa

stretch (aa 312–334) in the extracellular part of b3 could

interfere with integrin activation. An explanation for this

might be that the mutations in the extracellular part

change the conformation so drastically that the cyto-

plasmic tail also changes.

Information on possible regulatory domains in the

aIIb , the cytoplasmic tail is scarce. Deletion of the entire

cytoplasmic tail of aIIb or the conserved G991FFKR995

sequence, shifts the integrin into a high affinity state,

suggesting that the tail functions as a negative regulator,

locking the integrin in a low affinity state.40 Platelets can

be activated by introduction of a peptide mimicking the

membrane proximal region of aIIb (K989VGFFKR995 ).41

This activation was not caused by a direct effect on

aIIbb3 but by induction of thromboxane formation.

Another study points to two residues that seem to be

important for a proper conformation of the aIIb-

cytoplasmic tail.42 When these residues are mutated

(Pro998®Ala and Pro999

®Ala), the integrin changes

from a low affinity to a high affinity state.43 When wild-

type peptides of this region are incorporated in the

platelet, they inhibit the ligand binding to aIIbb3 .42 The

peptide containing the two Pro®Ala substitutions is

without any effect. An explanation is that the Pro

312 aIIbb3 AS TARGET AND GENERATOR OF SIGNALLING

Figure 2. The amino acid sequence of the aIIbb3-cytoplasmic tails and cytoplasmic tail binding proteins.

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substitutions completely change the conformation of the

aIIb-cytoplasmic tail from a very compact to a very loose

structure probably resulting in different interactions with

the b3-cytoplasmic tail.

Besides the positive regulation of aIIbb3 there are also

negative regulators of agonist-induced ligand binding.

Most of them do not affect the integrin itself, but

interfere with the signalling by the agonist receptor. The

most prominent are cAMP, formed after engagement of

prostanoid receptors with compounds such as prosta-

cylcin and cGMP, formed as a result of activation of

cytoplasmic guanylate cyclase by compounds such as

nitric oxide. After the rise in cAMP and cGMP, protein

kinase A (PKA) and protein kinase G (PKG) are

activated, respectively, and signalling to the integrin is

inhibited rather than the integrin itself. PKA and PKG

have a common substrate in platelets called vasodilator-

stimulated phosphoprotein (VASP).44 VASP is associated

with actin filaments and is also present in focal

adhesions.45 The phosphorylation state of this protein

correlated with the inhibition of platelet aggregation.45,46

Furthermore, the cAMP- and cGMP-mediated inhibition

of platelet aggregation was markedly reduced in platelets

of the VASP knock-out mouse, while the inhibition on

platelet secretion and the rise in intracellular Ca2+ was

not affected.47 Whether VASP interacts with aIIbb3

thereby changing the conformation into a low affinity

state or inhibits other processes needed for integrin

activation is not known.

One report suggests a direct negative effect on aIIbb3

by the small GTPase H-ras.48 Transfection of mutation-

ally activated H-ras into aIIbb3 expressing CHO cells,

suppressed the activation of the integrin. A similar

inhibition was seen with an effector kinase of H-ras, raf–

1. Both effects correlated with activation of the ERK/

MAP kinase pathway and were independent of Tyr-

phosphorylation of the b3-cytoplasmic tail. Also the

b3-endonexin-mediated activation of aIIbb3 was blocked

by H-ras.21 In platelets ras is present, but the subtype is

unknown. 49,50 It is activated by a-thrombin49,50 as is the

ERK/MAP kinase pathway.51,52 It appears contradictory

that a powerful activator of aIIbb3 also activates a

powerful inhibitory pathway of aIIbb3 . Zhang et al.53

have shown that another subtype of ras, R-ras, was an

activator of integrins. This activation, however, was not

a direct effect of R-ras on the integrin, but the result of

suppression of the H-ras/raf pathway.54 Thus, it is

important to know which subtypes of ras are present in

platelets.

Signalling generated by aIIbb3

A crucial step in the formation of a stable aggregate is

signal generation by the integrin aIIbb3 itself. This

outside-in signalling can be subdivided into ligand-

dependent and ligand-independent outside-in signalling.

This subdivision is based on an early observation by

Clark and co-workers55,56 who defined three phases in

Tyr-phosphorylation after platelet activation: (1) a first

phase induced by the agonist; (2) a second phase induced

by the integrin affinity change; and (3) a third phase

induced by ligand binding to the integrin, integrin

clustering and changes in avidity.

The cytoplasmic tails of aIIb and b3 do not contain

recognisable signalling sequences, such as kinase

domains and binding sites for phosphorylated proteins,

the SH2 and SH3 domains (Fig. 2). This suggest that

signalling involves proteins that interact directly or via a

linker protein with the integrin cytoplasmic tails. CIB

and b3-endonexin, also deprived of signalling sequences

such as kinase domains, could function as linker

proteins.

One of the kinases involved in ligand-independent

outside-in signalling is the Spleen tYrosine Kinase or in

short Syk. The activation of this Tyr-kinase requires both

an intact aIIb-cytoplasmic tail as well as an intact

b3-cytoplasmic tail.57 Syk activation does not depend on

a change in affinity of the integrin complex, but is

already triggered by receptor clustering in the absence of

a ligand29 and depends on autophosphorylation and

phosphorylation by the Tyr-kinase Src.57 Hence Syk

activation requires a change in avidity of the complex

rather than a change in affinity. Activation of Syk leads

to various responses. In CHO cells that express aIIbb3

and in platelets activation of Syk results in Tyr-

phosphorylation of the guanine nucleotide exchange

factor (GEF) Vav,58,59 which is the trigger for the

activation of this protein. Vav and its close homologue

Vav260,61 are responsible for the activation of three

members of the rho-family of GTPases, e.g. rho, rac and

cdc42.62– 66 These proteins are essential for cytoskeletal

reorganisation and the formation of filapodia (cdc42),

lamellapodia (rac) and actin stress-fibres (rho).67– 73 This

cytoskeletal reorganisation is essential for adhesion and

spreading to surface-coated fibrinogen (Fig. 3). Rho is

also involved in platelet shape change.74

Another protein that becomes Tyr-phosphorylated by

Syk is the adapter protein Cbl,58 which can function as a

linker protein for phosphatidylinositol (PI) 3-kinase.75

Shattil and co-workers suggested that Tyr-phosphory-

lated Cbl could link PI3-kinase to aIIbb3 . PI3-kinase is

an essential enzyme in the reorganisation of the cytoske-

leton.76,77 Hartwig and colleagues showed that an active

PI3-kinase is needed for stable interaction of a ligand

with aIIbb3 .76

Whether the role of Syk is really important for this is

a matter of debate. Adhesion studies performed with

platelets from a Syk null-mouse show no difference in

the number of platelets adhered to fibrinogen compared

to platelets of a normal mouse.78 Platelet spreading was

not measured but in view of the effect of Syk on

cytoskeletal reorganisation spreading may have been

impaired. When an inhibitor of Syk was used (picea-

tannol) adhesion was reduced.78 This discrepancy was

attributed to the lack of specificity of the inhibitor.

The kinase that supports ligand-dependent outside-in

signalling is the Focal Adhesion Kinase or in short FAK.

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Activation of this kinase depends only on an intact

b3-cytoplasmic tail.29,57,79 Activated FAK is essential for

the formation of focal adhesions. These are large multi-

protein complexes that are assembled on actin-stress

fibres at the spot where the cells undergo stable

interaction with fibrinogen. Phosphorylated FAK is a

docking site for Grb2 and Shc,80,81 both involved in the

activation of the small GTPase ras, and Src80 (Fig. 4).

FAK is also the site of interaction of the adapter

molecule Crk-Associated Substrate or Cas when this

protein becomes phosphorylated on Tyr-residues.80,82 – 84

In the phosphorylated state Cas serves as a docking

protein for various Tyr-kinases of the Src family of

kinases, such as Lyn, Fyn and Src itself.81 In platelets

only an interaction with Src could be demonstrated.85

Cas is also involved in cytoskeletal reorganisation.86

Taken together, this might be one of the mechanisms that

translocates the kinases and adapter proteins to the

cytoskeleton and localise them in the focal adhesions.

Interestingly, Cas also binds the GEF C3G which affects

the small GTPase rap1.87 This interaction may explain

the aIIbb3-mediated effects on rap1 activity.88

The phosphorylation of Cas is not dependent on

aIIbb3 , but the protein is dephosphorylated via a

mechanism that depends on ligand binding to aIIbb3 .85

This suggests that aIIbb3 regulates the translocation to

the cytoskeleton of the Src family of kinases and adapter

proteins involved in ras activation.

An alternative kinase for the FAK-induced signalling

is the Proline-rich tYrosine Kinase 2 (Pyk2), which is

also known as RAFTK, CAKb and CADTK. This

protein kinase is a close homologue of FAK with similar

consensus motifs in the central kinase domain, the

absence of SH2 and SH3 domains and also in respect to

proteins that bind to the phosphorylated form.59,89

Similar to FAK, Pyk2 can be responsible for cytoskeletal

reorganisation. In platelets Pyk2 is, like FAK, activated

in an aIIbb3-dependent and ligand-dependent manner.89

Also the time course of activation is similar to that of

FAK.89 This suggests that Pyk2 may be responsible for

some of the actions attributed to FAK. This is illustrated

by experiments with FAK knock-out mice.90 Cells from

these mice showed the formation of more and larger

focal adhesions. This leads to the hypothesis that Pyk2 is

the initiator of focal adhesion formation, with FAK

acting as a negative regulator.

An alternative integrin-regulated pathway for cytoske-

letal reorganisation involves the Ca2+ -dependent

cysteine- or thiol-protease calpain with m-calpain present

in platelets.91,92 Activation of calpain is seen after

aggregation of platelets or when platelets spread on

extracellular matrix proteins.93 – 95 After activation many

platelet proteins become a substrate for calpain. Among

them are cytoskeletal proteins such as talin and filamin,

but also signalling molecules such as PKC, Src and

FAK.92,96– 99 Furthermore, the b3-cytoplasmic tails are

cleaved at four places by calpain.100,101 Calpain mod-

ulates the late events of platelet-mediated clot retrac-

tion.102 Calpain activation in platelets is dependent on

aIIbb3 and inhibition of the protease blocks platelet

aggregation, secretion and platelet spreading,103 all

processes that depend on a proper cytoskeletal rearrange-

ment. In bovine aortic endothelial cells, Kulkarni et al.73

elegantly showed that inhibition of calpain abolished

cytoskeletal rearrangement and the subsequent spreading

of cells. This was the result of inhibition of rac and rho,

suggesting that calpain activity is upstream of these

small GTPases.

How the integrin regulates the activity of calpain is

still not known. It is possible that calpain interacts with

one of the integrin cytoplasmic tails, thereby coming in

close contact with substrates like Src and FAK. Fur-

thermore, integrins like aIIbb3 have been shown to

function as Ca2+ -channels.104,105 Interaction of calpain

with the integrin allows exposure of the protease to high

local Ca2+-concentrations. Also the interaction between

314 aIIbb3 AS TARGET AND GENERATOR OF SIGNALLING

Figure 3. Role of aIIbb3 in cytoskeletal reorganisation. TheaIIbb3-complex is involved in the activation of the Tyr-kinase Sykand of the Ca2+-dependent protease m-calpain. Syk becomesphosphorylated by Src and subsequently phosphorylates andactivates the guanine nucleotide exchange factor Vav. Both Vavand m-calpain can activate cdc42, rac and rho, members of therho-family of small GTPases. Activation of these small GTPasesleads to rearrangement of the cytoskeleton (L = ligand; aIIbb3* =exposed conformation of aIIbb3 ).

Figure 4. Multiple interactions in the focal adhesion plaque.Depicted is a simplified scheme of interactions that occur in thefocal adhesion plaque. When aIIbb3 becomes occupied by aligand the b3-chain of the integrin becomes phosphorylated onTyr-residues and also FAK becomes Tyr-phosphorylated, thetrigger for activation of FAK. This form of FAK interacts with actinstress fibres, the phosphorylated adaptor protein Cas and alsowith Shc and Grb2, which both function in the ras-pathway. Casinteracts with Src and with the guanine nucleotide exchangefactor C3G of the small GTPase rap1 (L = ligand; aIIbb3* =exposed conformation of aIIbb3 ).

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integrins and calreticulin regulates the Ca2+ -influx in

cells and cell adhesion, providing an alternative mecha-

nism of integrin-mediated calpain activation.106

The b3 can also serve as a docking site for adapter

proteins involved in the ras pathway. In the b3-cyto-

plasmic tail two NXXY-motifs (N744PLY747 and

N756ITY759 ) are present and form the so-called Integrin

Cytoplasmic tYrosine motif or ICY-motif. The Tyr-

residues in the ICY-motifs become phosphorylated after

ligand occupancy of aIIbb3 . Using Tyr-phosphorylated

peptides (Tyr747 and Tyr759 ) that mimic the b3-cyto-

plasmic tail (aa 740–762), Law et al.107 showed that Shc

bound to the Tyr759 and to the Tyr747/Tyr759-phosphory-

lated peptide, while Grb2 only bound to the dual-

phosphorylated peptide. Both proteins did not bind to the

non-phosphorylated peptide or the Tyr747-phosphory-

lated form. The Ser752®Pro substitution in the dual Tyr-

phosphorylated peptide completely blocked the binding

of Grb2 and reduced the binding of Shc substantially.

Several studies tried to identify domains in the

cytoplasmic regions of aIIbb3 involved in outside-in

signalling. In the platelets of a patient with the

Ser752®Pro mutation both Syk and FAK phosphoryla-

tion were absent,31,32 confirming that the phosphoryla-

tion and activation of these two kinases depend on an

intact b3-cytoplasmic tail. The activation of FAK

depends on the two ICY-motifs and on a region in the

membrane proximal part (aa 717–729).23,108 The latter

motif is the likely site for the interaction of FAK with the

b3 molecule. Mutations in either of the ICY-motifs or in

the membrane proximal region inhibits FAK phosphor-

ylation. In our study with SLO-permeabilised platelets

and peptides mimicking part of the b3 cytoplasmic tail,

we found that the region in b3 comprised of aa 749–756

completely blocked a-thrombin-induced phosphoryla-

tion of both Syk and FAK.109 A peptide mimicking the

region T755NITYRGT762 was ineffective. This shows

that the region E749ATSTFTN756 is involved in inside-

out as well as outside-in signalling. It is uncertain

whether Syk and FAK directly interact with b3 or via a

docking molecule. Interestingly, b3 contains a putative

FAK binding site (aa 717–729).23

Microinjection of the peptide E749ATSTFTN756 into

aIIbb3 expressing CHO cells that adhered to fibrinogen

resulted in complete de-adhesion of the cells and the

cells disappeared from the surface.109 The peptide

mimicking the region T755NITYRGT762 failed to de-

adhere the cells, but the focal adhesion plaques became

more dispersed. This again shows the great importance

of the region E749–N756 in the formation of focal

adhesion plaques. It also shows that in adherent cells the

formation and degradation of focal adhesions is a

continuous process. The effect of the peptide T755–T762

indicates that b3-endonexin binding to b3 may be

involved in focal adhesion formation.

Replacement of the two Tyr-residue by Phe in a mouse

model did not interfere with ligand binding to aIIbb3 , but

aggregation and clot-retraction were impaired.110 Also

the tendency to re-bleed was high in these animals. The

explanation for these observations is that the formed

aggregate is very unstable owing to a defect in the

cytoskeletal reorganisation. An interesting detail is that

the Tyr in the N756ITY759 motif is essential for

b3-endonxin binding to b3 ,38 suggesting that b3-endo-

nexin is involved in outside-in signalling.

In addition to these pathways in outside-in signalling a

fascinating new mechanism for integrin signalling comes

into focus (reviewed in Woods and Couchman111 ). This

mechanism does not involve binding of cytoplasmic

proteins to the integrin cytoplasmic tails, but is based on

sideways interaction of the integrin with other transmem-

brane proteins. One of them is the proteoglycan synde-

can–4 which regulates cytoskeletal reorganisation112– 114

and associates and activates protein kinases such as

PKCa.115 –117 This protein can bind to integrins and co-

localises with integrins in focal adhesions.112–114,11 8

Other proteins that interact with integrins in the plane

of the membrane are members of the transmembrane–4

superfamily (TM4SF) or tetraspannins. These proteins

signal to the cytoskeleton and protein kinases, such as

FAK119 and PKC.25 Several members of the TM4SF are

present on platelets, e.g. CD9, CD63 and CD151 (also

known as PETA–3).120– 123 CD9 and CD151, interact

with aIIbb3 .121,123–125 CD9 is co-localised with aIIbb3 in

the leading edge of the lamellapodia of adhering cells but

not in focal adhesions.126 A similar distribution is seen

for CD151.125 CD151 does not change the affinity or

avidity of aIIbb3 , but increases cell–cell adhesion,125

suggesting that CD151 is a component of the aIIbb3-

signalling complex.

Whether syndecan–4 and TM4SF members besides

CD151 contribute to aIIbb3 signalling is not known to date.

From these considerations it is clear that the main

target of outside-in signalling is the cytoskeleton leading

to changes in aIIbb3 avidity due to receptor clustering

and stabilisation of ligand binding. However, there are

many other signalling processes positively or negatively

affected by aIIbb3 , such as the activation of the small

GTPase rap1,88 PI-metabolism107,127 and the activation

of ERK2128 to name but a few. Whether this is all due to

the effects of aIIbb3 on the cytoskeleton remains to be

investigated.

Clinical implications of signalling defects

Defects in inside-out and outside-in signalling may

account for part of the aggregation defects in platelets

with a (sub)normal expression of aIIbb3 . Furthermore,

differences in inside-out and outside-in signalling may

account for subjects with increased thrombotic risk.

One of the patients with a defective inside-out and

outside-in signalling has already been mentioned. This

patient has a substitution of Ser752®Pro in b3 , but the

amount of aIIbb3 expressed on the platelet membrane is

only slightly abnormal.31,32 Recently it was shown that

the signals that in normal platelets change the affinity of

the aIIbb3 are also operational in the platelets of this

patient. The ligand binding to the integrin is very

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unstable owing to the defects in outside-in signalling.129

Another patient has been described with defects in

inside-out and outside-in signalling owing to a truncated

cytoplasmic tail of b3 (b3 724).130

Another class of patients is that comprising those with

defects in the signalling pathways that lead to the affinity

change of the integrin. Examples of this class of patients

are (1) a patient that has a decreased level of PLCb2 and

has an impaired aggregation response131 and (2) a patient

who has decreased levels of the Gq-protein and suffers

from a mild bleeding disorder.132

Recently, a link between differences in outside-in

signalling and an increased thrombotic risk has been

described. Platelets of subjects carrying the PLA2

polymorphism in b3 (substitution of Leu33®Pro, located

in the Cys-rich region of b3 ) respond to lower agonist

concentrations with aggregation and a-granule secre-

tion.132,133 Furthermore, in some but not all studies, the

PLA2 polymorphism is related to an increased incidence

of ischemic vascular disease. Platelets with the PLA2

polymorphism also show an increased adhesion and

better spreading on a fibrinogen surface, spread better on

fibrinogen and show an increased clot retraction. Surpris-

ingly, the binding of soluble fibrinogen is not changed.

These observations point to a normal inside-out signal-

ling combined with enhanced outside-in signalling. The

group of Bray found that one of the key players in

outside-in signalling, FAK, showed increased Tyr-phos-

phorylation.134 Thus, more FAK was activated after

ligation of aIIbb3 in the cells with the PLA2 polymor-

phism. Now the question remains how an extracellular

mutation can affect intracellular processes. Such a

phenomenon was already observed by Baker et al. (see

above).39

In conclusion

It may be clear that there is a large body of information

regarding inside-out and outside-in signalling. However,

it is still a long way to unravel all mechanisms and

signalling routes involved. Exciting new mechanisms for

integrin-mediated signalling are found, which further

contribute to the complexity of aIIbb3 control. It will be

a challenge to use this basic information to explain

poorly understood platelet aggregation defects.

Acknowledgements

PEHML is supported by The Netherlands Organisation for ScientificResearch (grant 902–26–186), JWNA is supported by The Netherlands

Thrombosis Foundation and GvW is a research fellow of the CatharijneFoundation and supported by the Dirk Zwager-Assink Foundation.

References

1. Loftus J C, Liddington R C. Cell adhesion in vascular biology.New insights into integrin–ligand interaction. J Clin Invest 1997;

99: 2302–06.2. Savage B, Almus-Jacobs F, Ruggeri Z M. Specific synergy of

multiple substrate–receptor interactions in platelet thrombus

formation under flow. Cell 1998; 94: 657– 66.

3. Guinebault C, Payrastre B, Sultan C, Mauco G, Breton M, Levy-Toledano S et al. Tyrosine kinases and phosphoinositide metabo-lism in thrombin-stimulated human platelets. Biochem J 1993;

292: 851–6.4. Kahn M L, Zheng Y W, Huang W, Bigornia V, Zeng D W, Moff

S et al. A dual thrombin receptor system for platelet activation.

Nature 1998; 394: 690– 94.5. Offermanns S, Toombs C F, Hu Y H, Simon M I. Defective

platelet activation in Gaq-deficient mice. Nature 1997; 389:

183–6.6. van Willigen G, Hers I, Gorter G, Akkerman J-W N. Exposure of

ligand-binding sites on platelet integrin aIIb/b3 by phosphoryla-tion of the b3 subunit. Biochem J 1996; 314: 769–79.

7. Parise L V, Criss A B, Nannizzi L, Wardell M R. Glycoprotein IIIais phosphorylated in intact human platelets. Blood 1990; 75:2363– 68.

8. Lerea K M, Cordero K P, Sakariassen K S, Kirk R I, Fried V A.Phosphorylation sites in the integrin b3 cytoplasmic domain inintact platelets. J Biol Chem 1999; 274: 1914–19.

9. van Willigen G, Akkerman J-W N. Regulation of glycoproteinIIB/IIIA exposure on platelets stimulated with a-thrombin. Blood

1992; 79: 82–90.

10. Shattil S J, Brass L F. Induction of the fibrinogen receptor onhuman platelets by intracellular mediators. J Biol Chem 1987;262: 992–1000.

11. van Willigen G, Akkerman J-W N. Protein kinase C and cyclic

AMP regulate reversible exposure of binding sites for fibrinogenon the glycoprotein IIB–IIIA complex of human platelets.Biochem J 1991; 273: 115– 20.

12. Hers I, Donath J, van Willigen G, Akkerman J-W. Differentialinvolvement of tyrosine and serine/threonine kinases in plateletintegrin aIIbb3 exposure. Arterioscler Thromb Vasc Biol 1998; 18:

404–14.13. Hers I, Donath J, Litjens P E M H, van Willigen G, Akkerman

J-W N. Inhibition of platelet integrin aIIbb3 by peptides that

interfere with protein kinases and the b3-tail. Arterioscler Thromb

Vasc Biol 2000; 20: 1651– 60.14. Daniel J L, Dangelmaier C, Smith J B. Evidence for a role for

tyrosine phosphorylation of phospholipase Cg2 in collagen-induced platelet cytosolic calcium mobilization. Biochem J 1994;302: 617–22.

15. Blake R A, Schieven G L, Watson S P. Collagen stimulates

tyrosine phosphorylation of phospholipase C-g2 but not phospho-lipase C-g1 in human platelets. FEBS Lett 1994; 353: 212–16.

16. Asselin J, Gibbins J M, Achison M, Lee Y H, Morton L F,

Farndale R W et al. Collagen-like peptide stimulates tyrosinephosphorylation of syk and phospholipase Cg2 in plateletsindependent of the integrin a2b1 . Blood 1997; 89: 1235– 42.

17. Blystone S D, Williams M P, Slater S E, Brown E J. Requirementof integrin b3 tyrosine 747 for b3 tyrosine phosphorylation andregulation of avb3 avidity. J Biol Chem 1997; 272: 28757–61.

18. Law D A, Nannizzi-Alaimo L, Phillips D R. Outside-in integrinsignal transduction–aIIbb3-(GP IIb–IIIa) tyrosine phosphoryla -tion induced by platelet aggregation. J Biol Chem 1996; 271:

10811–15.19. Naik U P, Patel P M, Parise L V. Identification of a novel calcium-

binding protein that interacts with the integrin aIIb cytoplasmicdomain. J Biol Chem 1997; 272: 4651– 54.

20. Shattil S J, O’Toole T, Eigenthaler M, Thon V, Williams M,Babior B M et al. b3-endonexin, a novel polypeptide that interactsspecifically with the cytoplasmic tail of the integrin b3 subunit. J

Cell Biol 1995; 131: 807–16.21. Kashiwagi H, Schwartz M A, Eigenthaler M, Davis K A,

Ginsberg M H, Shattil S J. Affinity modulation of platelet

integrin aIIbb3 by b3-endonexin, a selective binding partner ofthe b3 integrin cytoplasmic tail. J Cell Biol 1997; 137:1433– 43.

22. Bennett J S, Zigmond S, Vilaire G, Cunningham M E, Bednar B.The platelet cytoskeleton regulates the affinity of the integrinaI Ibb3 for fibrinogen. J Biol Chem 1999; 274: 25301–07.

23. Lyman S, Gilmore A, Burridge K, Gidwitz S, White G C.

Integrin-mediated activation of focal adhesion kinase is inde-pendent of focal adhesion formation or integrin activation.Studies with activated and inhibitory b3 cytoplasmic domain

mutants. J Biol Chem 1997; 272: 22538–47.24. Reddy K B, Gascard P, Price M G, Negrescu E V, Fox J E B.

Identification of an interaction between the m-band protein

skelemin and beta-integrin subunits. Colocalization of a

316 aIIbb3 AS TARGET AND GENERATOR OF SIGNALLING

-45-

Page 46: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

skelemin-like protein with b1- and b3-integrins in non-musclecells. J Biol Chem 1998; 273: 35039–47.

25. Hemler M E. Integrin associated proteins. Curr Opin Cell Biol

1998; 10: 578–85.26. Knezevic I, Leisner T M, Lam S C T. Direct binding of the

platelet integrin aIIbb3 (GPIIb–IIIa) to talin–Evidence that

interaction is mediated through the cytoplasmic domains of bothaI Ib and b3 . J Biol Chem 1996; 271: 16416–21.

27. Patil S, Jedsadayanmata A, Wencel-Drake J D, Wang W,

Knezevic I, Lam S C T. Identification of a talin-binding site in theintegrin b3 subunit distinct from the NPLY regulatory motif ofpost-ligand binding functions–The talin N-terminal head domaininteracts with the membrane-proximal region of the b3 cyto-

plasmic tail. J Biol Chem 1999; 274: 28575–83.28. Schootemeijer A, van Willigen G, Van der Vuurst H, Tertoolen L

G J, De Laat S W, Akkerman J-W N. Lateral mobility of integrin

aI Ibb3 (glycoprotein IIb/IIIa) in the plasma membrane of a humanmegakaryocyte. Thromb Haemost 1997; 77: 143–49.

29. Hato T, Pampori N, Shattil S J. Complementary roles for receptor

clustering and conformational change in the adhesive andsignaling functions of integrin aI Ibb3 . J Cell Biol 1998; 141:1685– 95.

30. Ylanne J, Huuskonen J, O’Toole T E, Ginsberg M H, Virtanen I,Gahmberg C G. Mutation of the cytoplasmic domain of theintegrin b3 subunit. Differential effects on cell spreading,recruitment to adhesion plaques, endocytosis, and phagocytosis. J

Biol Chem 1995; 270: 9550–07.31. Chen Y-P, Djaffar I, Pidard D, Steiner B, Cieutat A-M, Caen J P

et al. Ser–752®Pro mutation in the cytoplasmic domain of

integrin b3 subunit and defective activation of platelet integrinaI Ibb3 (glycoprotein IIb–IIIa) in a variant of Glanzmannthrombasthenia. Proc Natl Acad Sci USA 1992; 89: 10169–73.

32. Chen Y-P, O’Toole T E, Ylanne J, Rosa J-P, Ginsberg M H. Apoint mutation in the integrin b3 cytoplasmic domain (S752

®P)

impairs bidirectional signaling through aIIbb3 (platelet glycopro-

tein IIb–IIIa). Blood 1994; 84: 1857– 65.33. O’Toole T E, Katagiri Y, Faull R J, Peter K, Tamura R, Quaranta

V et al. Integrin cytoplasmic domains mediate inside-out signal

transduction. J Cell Biol 1994; 124: 1047– 59.34. Kieffer N, Melchior C, Guinet J M, Michels S, Gouon V, Bron N.

Serine752 in the cytoplasmic domain of the b3 integrin subunit isnot required for avb3 postreceptor signaling events. Cell Adhes

Commun 1996; 4: 25–39.35. Perrault C, Mekrache M, Schoevaert D, Kieffer N, Melchior C,

Warszawski J et al. Ser752 mutation to Pro or Ala in the b3

integrin subunit differentially affects the kinetics of cell spreadingto von Willebrand factor and fibrinogen. Cell Adhes Commun

1998; 6: 335– 48.

36. Liu K Y, Timmons S, Lin Y Z, Hawiger J. Identification of afunctionally important sequence in the cytoplasmic tail of integrinb3 by using cell-permeable peptide analogs. Proc Natl Acad Sci

USA 1996; 93: 11819–24.37. Zhang L, Torgerson T R, Liu X Y, Timmons S, Colosia A D,

Hawiger J et al. Preparation of functionally active cell-permeable

peptides by single-step ligation of two peptide modules. Proc

Natl Acad Sci USA 1998; 95: 9184–89.38. Eigenthaler R, Hofferer L, Shattil S J, Ginsberg M H. A conserved

sequence motif in the integrin b3 cytoplasmic domain is required

for its specific interaction with b3-endonexin. J Biol Chem 1997;272: 7693– 98.

39. Baker E K, Tozer E C, Pfaff M, Shattil S J, Loftus J C,

Ginsberg M H. A genetic analysis of integrin function: Glanz-mann thrombasthenia in vitro. Proc Natl Acad Sci USA 1997; 94:1973–78.

40. O’Toole T E, Mandelman D, Forsyth J, Shattil S J, Plow E F,Ginsberg M H. Modulation of the affinity of integrin aIIbb3

(GPIIb–IIIa) by the cytoplasmic domain of aI Ib . Science 1991;

254: 845–47.41. Stephens G, O’Luanaigh N, Reilly D, Harriott P, Walker B,

Fitzgerald D et al. A sequence within the cytoplasmic tail ofGpIIb independently activates platelet aggregation and thrombox-

ane synthesis. J Biol Chem 1998; 273: 20317–22.42. Vinogradova O, Haas T, Plow E F, Qin J. A structural basis for

integrin activation by the cytoplasmic tail of the aI Ib-subunit.

Proc Natl Acad Sci USA 2000; 97: 1450–55.43. Leisner T M, Wencel-Drake J D, Wang W, Lam S C. Bidirectional

transmembrane modulation of integrin aIIbb3 conformations. J

Biol Chem 1999; 274: 12945–49.

44. Butt E, Abel K, Krieger M, Palm D, Hoppe V, Hoppe J et al.

cAMP-and cGMP-dependent protein kinase phosphorylation sitesof the focal adhesion vasodilator-stimulated phosphoprotei n(VASP) in vitro and in intact human platelets. J Biol Chem 1994;269: 14509–17.

45. Reinhard M, Halbrugge M, Scheer U, Wiegand C, Jockusch B M,

Walter U. The 46/50 kDa phosphoprotein VASP purified fromhuman platelets is a novel protein associated with actin filamentsand focal contacts. EMBO J 1992; 11: 2063–70.

46. Horstrup K, Jablonka B, Honig-Liedl P, Just M, Kochsiek K,Walter U. Phosphorylation of focal adhesion vasodilator-stimu-lated phosphoprotein at Ser157 in intact human platelets corre-lates with fibrinogen receptor inhibition. Eur J Biochem 1994;

225: 21–7.47. Aszodi A, Pfeifer A, Ahmad M, Glauner M, Zhou X H, Ny L et al.

The vasodilator-stimulated phosphoprotein (VASP) is involved in

cGMP- and cAMP-mediated inhibition of agonist-induced plate-let aggregation, but is dispensable for smooth muscle function.EMBO J 1999; 18: 37– 48.

48. Hughes P E, Renshaw M W, Pfaff M, Forsyth J, Keivens V M,Schwartz M A et al. Suppression of integrin activation: a novelfunction of a Ras/Raf-initiated MAP kinase pathway. Cell 1997;

88: 521– 30.49. Wolthuis R M, Franke B, Van Triest M, Bauer B, Cool R H,

Camonis J H et al. Activation of the small GTPase Ral inplatelets. Mol Cell Biol 1998; 18: 2486– 91.

50. Shock D D, He K, Wencel-Drake J D, Parise L V. Ras activationin platelets after stimulation of the thrombin receptor, thrombox-ane A2 receptor or protein kinase C. Biochem J 1997; 321:

525–30.51. van Willigen G, Gorter G, Akkerman J W. Thrombopoietin

increases platelet sensitivity to a-thrombin via activation of the

ERK2-cPLA2 pathway. Thromb Haemost 2000; 83: 610–16.52. Papkoff J, Chen R-H, Blenis J, Forsman J. p42 Mitogen-

activated protein kinase and p90 ribosomal S6 kinase are

selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation. Mol Cell Biol 1994;14: 463–72.

53. Zhang Z, Vuori K, Wang H, Reed J C, Ruoslahti E. Integrinactivation by R-ras. Cell 1996; 85: 61–9.

54. Sethi T, Ginsberg M H, Downward J, Hughes P E. The smallGTP-binding protein R-Ras can influence integrin activation by

antagonizing a Ras/Raf-initiated integrin suppression pathway.Mol Biol Cell 1999; 10: 1799–809.

55. Clark E A, Shattil S J, Brugge J S. Regulation of protein tyrosine

kinases in platelets. Trends Biochem Sci 1994; 19: 464– 69.56. Clark E A, Shattil S J, Ginsberg M H, Bolen J, Brugge J S.

Regulation of the protein tyrosine kinase pp72syk by platelet

agonists and the integrin aIIbb3 . J Biol Chem 1994; 269:28859– 64.

57. Gao J, Zoller K E, Ginsberg M H, Brugge J S, Shattil S J.

Regulation of the pp72syk protein tyrosine kinase by plateletintegrin aI Ibb3 . EMBO J 1997; 16: 6414–25.

58. Miranti C K, Leng L, Maschberger P, Brugge J S, Shattil S J.

Identification of a novel integrin signaling pathway involving thekinase Syk and the guanine nucleotide exchange factor Vav1.Curr Biol 1998; 8: 1289–99.

59. Cichowski K, Brugge J S, Brass L F. Thrombin receptor

activation and integrin engagement stimulate tyrosine phosphor-ylation of the proto-oncogene product, p95vav, in platelets. J Biol

Chem 1996; 271: 7544– 50.

60. Schuebel K E, Bustelo X R, Nielsen D A, Song B J, Barbacid M,Goldman D et al. Isolation and characterization of murine vav2,a member of the vav family of proto-oncogenes. Oncogene 1996;

13: 363–71.61. Cerione R A, Zheng Y. The Dbl family of oncogenes. Curr Opin

Cell Biol 1996; 8: 216–22.

62. Han J, Das B, Wei W, Van Aelst L, Mosteller R D, Khosravi-FarR et al. Lck regulates Vav activation of members of the Rhofamily of GTPases. Mol Cell Biol 1997; 17: 1346– 53.

63. Crespo P, Schuebel K E, Ostrom A A, Gutkind J S, Bustelo X R.

Phosphotyrosine-dependent activation of Rac–1 GDP/GTPexchange by the vav proto-oncogene product. Nature 1997; 385:169–72.

64. Olson M F, Pasteris N G, Gorski J L, Hall A. Faciogenitaldysplasia protein (FGD1) and Vav, two related proteins requiredfor normal embryonic development, are upstream regulators of

Rho GTPases. Curr Biol 1996; 6: 1628– 33.

PLATELETS 317

-46-

Page 47: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

65. Schuebel K E, Movilla N, Rosa J L, Bustelo X R. Phosphoryla-tion-dependent and constitutive activation of Rho proteins bywild-type and oncogenic Vav–2. EMBO J 1998; 17:

6608– 21.66. Abe K, Rossman K L, Liu B, Ritola K D, Chiang D, Campbell

S L et al. Vav2 is an activator of Cdc42, Rac1, and RhoA. J

Biol Chem 2000; 275: 10141–49.67. Hotchin N A, Hall A. The assembly of integrin adhesion

complexes requires both extracellular matrix and intracellular

rho/rac GTPases. J Cell Biol 1995; 131: 1857– 65.68. Clark E A, King W G, Brugge J S, Symons M, Hynes R O.

Integrin-mediated signals regulated by members of the rhofamily of GTPases. J Cell Biol 1998; 142: 573–86.

69. Price L S, Leng J, Schwartz M A, Bokoch G M. Activation ofRac and Cdc42 by integrins mediates cell spreading. Mol Biol

Cell 1998; 9: 1863–71.

70. Nobes C, Hall A. Regulation and function of the Rho subfamilyof small GTPases. Curr Opin Genet Dev 1994; 4: 77– 81.

71. Nobes C D, Hall A. Rho, rac, and cdc42 GTPases regulate the

assembly of multimolecular focal complexes associated with actinstress fibers, lamellipodia, and filopodia. Cell 1995; 81: 53–62.

72. Machesky L M, Hall A. Role of actin polymerization and

adhesion to extracellular matrix in Rac- and Rho-inducedcytoskeletal reorganization. J Cell Biol 1997; 138: 913–26.

73. Kulkarni S, Saido T C, Suzuki K, Fox J E. Calpain mediatesintegrin-induced signaling at a point upstream of Rho family

members. J Biol Chem 1999; 274: 21265–75.74. Bauer M, Retzer M, Wilde J I, Maschberger P, Essler M, Aepfel-

bacher M et al. Dichotomous regulation of myosin phosphoryla-

tion and shape change by Rho-kinase and calcium in intact humanplatelets. Blood 1999; 94: 1665–72.

75. Saci A, Pain S, Rendu F, Bachelot-Loza C. Fc receptor-mediated

platelet activation is dependent on phosphatidylinositol 3-kinaseactivation and involves p120Cbl. J Biol Chem 1999; 274:1898– 904.

76. Kovacsovics T J, Bachelot C, Toker A, Vlahos C J, Duckworth B,Cantley L C et al. Phosphoinositide 3-kinase inhibition sparesactin assembly in activating platelets but reverses platelet

aggregation. J Biol Chem 1995; 270: 11358–66.77. Hartwig J H, Kung S, Kovacsovics T, Janmey P A, Cantley L C,

Stossel T P et al. D3 phosphoinositides and outside-in integrinsignaling by glycoprotein IIb–IIIa mediate platelet actin assem-

bly and filopodial extension induced by phorbol 12-myristate13-acetate. J Biol Chem 1996; 271: 32986–93.

78. Law D A, Nannizzi-Alaimo L, Ministri K, Hughes P E, Forsyth

J, Turner M et al. Genetic and pharmacological analyses of Sykfunction in aIIbb3 signaling in platelets. Blood 1999; 93:2645– 52.

79. Leong L, Hughes P E, Schwartz M A, Ginsberg M H, Shattil S J.Integrin signaling: roles for the cytoplasmic tails of aIIbb3 in thetyrosine phosphorylation of pp125FA K . J Cell Sci 1995; 108:

3817– 25.80. Schlaepfer D D, Hunter T. Focal adhesion kinase overexpression

enhances ras-dependent integrin signaling to ERK2/mitogen-

activated protein kinase through interactions with and activationof c-Src. J Biol Chem 1997; 272: 13189–95.

81. Zhu T, Goh E L, LeRoith D, Lobie P E. Growth hormonestimulates the formation of a multiprotein signaling complex

involving p130Cas and CrkII. Resultant activation of c-JunN-terminal kinase/stress-activated protein kinase (JNK/SAPK). JBiol Chem 1998; 273: 33864–75.

82. Polte T R, Hanks S K. Interaction between focal adhesion kinaseand Crk-associated tyrosine kinase substrate p130Cas. Proc Natl

Acad Sci USA 1995; 92: 10678–82.

83. Polte T R, Hanks S K. Complexes of focal adhesion kinase (FAK)

and Crk-associated substrate (p130Cas) are elevated in cytoskele-ton-associated fractions following adhesion and Src transforma-

tion. Requirements for Src kinase activity and FAK proline-richmotifs. J Biol Chem 1997; 272: 5501– 09.

84. Harte M T, Hildebrand J D, Burnham M R, Bouton A H, ParsonsJ T. p130C as, a substrate associated with v-Src and v-Crk,

localizes to focal adhesions and binds to focal adhesion kinase. JBiol Chem 1996; 271: 13649–55.

85. Ohmori T, Yatomi Y, Inoue K, Satoh K, Ozaki Y. Tyrosine

dephosphorylation, but not phosphorylation, of p130C as isdependent on integrin aIIbb3-mediated aggregation in platelets:implication of p130C as involvement in pathways unrelated to

cytoskeletal reorganization. Biochemistry 2000; 39: 5797–807.

86. Honda H, Oda H, Nakamoto T, Honda Z, Sakai R, Suzuki T et al.Cardiovascular anomaly, impaired actin bundling and resistanceto Src-induced transformation in mice lacking p130C as. Nat Genet

1998; 19: 361–65.87. Kirsch K H, Georgescu M M, Hanafusa H. Direct binding of

p130C as to the guanine nucleotide exchange factor C3G. J Biol

Chem 1998; 273: 25673–79.88. Franke B, Van Triest M, de Bruijn K M, van Willigen G,

Nieuwenhuis H K, Negrier C et al. Sequential regulation of the

small GTPase Rap1 in human platelets. Mol Cell Biol 2000; 20:779–85.

89. Ohmori T, Yatomi Y, Asazuma N, Satoh K, Ozaki Y. Involvementof proline-rich tyrosine kinase 2 in platelet activation: tyrosine

phosphorylation mostly dependent on aIIbb3 integrin and proteinkinase C, translocation to the cytoskeleton and association withShc through Grb2. Biochem J 2000; 347: 561–69.

90. Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N et

al. Reduced cell motility and enhanced focal adhesion contactformation in cells from FAK-deficient mice. Nature 1995; 377:

539–44.91. Saido T C, Suzuki H, Yamazaki H, Tanoue K, Suzuki K. In situ

capture of m-calpain activation in platelets. J Biol Chem 1993;

268: 7422– 26.92. Schoenwaelder S M, Kulkarni S, Salem H H, Imajoh-Ohmi S,

Yamao-Harigaya W, Saido T C et al. Distinct substrate specific-ities and functional roles for the 78- and 76-kDa forms of

m-calpain in human platelets. J Biol Chem 1997; 272:24876– 84.

93. Fox J E, Taylor R G, Taffarel M, Boyles J K, Goll D E. Evidence

that activation of platelet calpain is induced as a consequence ofbinding of adhesive ligand to the integrin, glycoprotein IIb–IIIa.J Cell Biol 1993; 120: 1501– 07.

94. Fox J E, Reynolds C C, Phillips D R. Calcium-dependen tproteolysis occurs during platelet aggregation. J Biol Chem 1983;258: 9973– 81.

95. Yuan Y, Dopheide S M, Ivanidis C, Salem H H, Jackson S P.Calpain regulation of cytoskeletal signaling complexes in vonWillebrand factor-stimulated platelets. Distinct roles for glyco-

protein Ib–V–IX and glycoprotein IIb–IIIa (integrin aIIbb3 ) invon Willebrand factor-induced signal transduction. J Biol Chem

1997; 272: 21847–54.96. Kishimoto A, Mikawa K, Hashimoto K, Yasuda I, Tanaka S,

Tominaga M et al. Limited proteolysis of protein kinase Csubspecies by calcium-dependent neutral protease (calpain). J

Biol Chem 1989; 264: 4088–92.

97. Tapley P M, Murray A W. Evidence that treatment of plateletswith phorbol ester causes proteolytic activation of Ca2+ -activated,phospholipid-dependent protein kinase. Eur J Biochem 1985;

151: 419–23.98. Oda A, Druker B J, Ariyoshi H, Smith M, Salzman E W. pp60src

is an endogenous substrate for calpain in human blood platelets.

J Biol Chem 1993; 268: 12603–08.99. Cooray P, Yuan Y, Schoenwaelder S M, Mitchell C A, Salem H H,

Jackson S P. Focal adhesion kinase (pp125FA K ) cleavage and

regulation by calpain. Biochem J 1996; 318: 41–7.100. Du X, Saido T C, Tsubuki S, Indig F E, Williams M J, Ginsberg M

H. Calpain cleavage of the cytoplasmic domain of the integrin b3

subunit. J Biol Chem 1995; 270: 26146–51.

101. Pfaff M, Du X, Ginsberg M H. Calpain cleavage of integrin b

cytoplasmic domains. FEBS Lett 1999; 460: 17–22.102. Schoenwaelder S M, Yuan Y, Cooray P, Salem H H, Jackson S P.

Calpain cleavage of focal adhesion proteins regulates thecytoskeletal attachment of integrin aIIbb3 (platelet glycoproteinIIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem

1997; 272: 1694–702.103. Croce K, Flaumenhaft R, Rivers M, Furie B, Furie B C, Herman

I M et al. Inhibition of calpain blocks platelet secretion,

aggregation, and spreading. J Biol Chem 1999; 274:36321– 27.

104. Brass L F. Ca2+ homeostasis in unstimulated platelets. J Biol

Chem 1984; 259: 12563–70.

105. Brass L F. Ca2+ transport across the platelet plasma membrane. Arole for membrane glycoproteins IIB and IIIA. J Biol Chem 1985;260: 2231– 36.

106. Coppolino M G, Woodside M J, Demaurex N, Grinstein S, StArnaud R, Dedhar S. Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature 1997; 386:

843–47.

318 aIIbb3 AS TARGET AND GENERATOR OF SIGNALLING

-47-

Page 48: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

107. Torti M, Bertoni A, Sinigaglia F, Balduini C, Payrastre B,Plantavid M et al. The platelet cytoskeleton regulates theaggregation-dependent synthesis of phosphatidylinosito l

3,4-bisphosphate induced by thrombin. FEBS Lett 2000; 466:355–58.

108. Tahiliani P D, Singh L, Auer K L, LaFlamme S E. The role of

conserved amino acid motifs within the integrin b3 cytoplasmicdomain in triggering focal adhesion kinase phosphorylation. J

Biol Chem 1997; 272: 7892–98.

109. Litjens P E M H, de Jong-Donath J, Ylanne J, Akkerman J-W N,van Willigen G. The role of two domains in the b3-cytoplasmictail of the integrin aI Ibb3 on integrin function. Thromb Haemost

2000; 82 (Suppl): 248.

110. Law D A, DeGuzman F R, Heiser P, Ministri-Madrid K, KilleenN, Phillips D R. Integrin cytoplasmic tyrosine motif is requiredfor outside-in aI Ibb3 signalling and platelet function. Nature

1999; 401: 808–11.111. Woods A, Couchman J R. Integrin modulation by lateral

association. J Biol Chem 2000; 275: 24233– 6.

112. Longley R L, Woods A, Fleetwood A, Cowling G J, Gallagher JT, Couchman J R. Control of morphology, cytoskeleton andmigration by syndecan–4. J Cell Sci 1999; 112: 3421– 31.

113. Echtermeyer F, Baciu P C, Saoncella S, Ge Y, Goetinck P F.Syndecan–4 core protein is sufficient for the assembly of focaladhesions and actin stress fibers. J Cell Sci 1999; 112:3433– 41.

114. Saoncella S, Echtermeyer F, Denhez F, Nowlen J K, Mosher D F,Robinson S D et al. Syndecan–4 signals cooperatively withintegrins in a Rho-dependent manner in the assembly of focal

adhesions and actin stress fibers. Proc Natl Acad Sci USA 1999;96: 2805–10.

115. Oh E S, Woods A, Couchman J R. Syndecan–4 proteoglycan

regulates the distribution and activity of protein kinase C. J Biol

Chem 1997; 272: 8133– 36.116. Oh E S, Woods A, Couchman J R. Multimerization of the

cytoplasmic domain of syndecan–4 is required for its ability toactivate protein kinase C. J Biol Chem 1997; 272: 11805–11.

117. Oh E S, Woods A, Lim S T, Theibert A W, Couchman J R.

Syndecan–4 proteoglycan cytoplasmic domain and phosphatidy -linositol 4,5-bisphosphate coordinately regulate protein kinase Cactivity. J Biol Chem 1998; 273: 10624–29.

118. Woods A, Couchman J R. Syndecan 4 heparan sulfate proteogly-

can is a selectively enriched and widespread focal adhesioncomponent. Mol Biol Cell 1994; 5: 183– 92.

119. Berditchevski F, Odintsova E. Characterization of integrin–

tetraspanin adhesion complexes: role of tetraspanins in integrinsignaling. J Cell Biol 1999; 146: 477–92.

120. Hasegawa H, Utsunomiya Y, Kishimoto K, Yanagisawa K, Fujita

S. SFA–1, a novel cellular gene induced by human T-cellleukemia virus type 1, is a member of the transmembrane 4superfamily. J Virol 1996; 70: 3258– 63.

121. Slupsky J R, Seehafer J G, Tang S C, Masellis-Smith A, Shaw AR. Evidence that monoclonal antibodies against CD9 antigeninduce specific association between CD9 and the platelet

glycoprotein IIb–IIIa complex. J Biol Chem 1989; 264:12289– 93.

122. Nishibori M, Cham B, McNicol A, Shalev A, Jain N, Gerrard JM.

The protein CD63 is in platelet dense granules, is deficient in apatient with Hermansky-Pudlak syndrome, and appears identicalto granulophysin. J Clin Invest 1993; 91: 1775–82.

123. Indig F E, Diaz-Gonzalez F, Ginsberg M H. Analysis of thetetraspanin CD9-integrin aIIbb3 (GPIIb–IIIa) complex in plateletmembranes and transfected cells. Biochem J 1997; 327:

291–98.124. Slupsky J R, Kamiguti A S, Rhodes N P, Cawley J C, Shaw A

R, Zuzel M. The platelet antigens CD9, CD42 and integrinalpha IIb beta IIIa can be topographically associated and

transduce functionally similar signals. Eur J Biochem 1997;244: 168–75.

125. Fitter S, Sincock P M, Jolliffe C N, Ashman L K. Transmembrane

4 superfamily protein CD151 (PETA–3) associates with b1 andaI Ibb3 integrins in haemopoietic cell lines and modulates cell–cell adhesion. Biochem J 1999; 338: 61–70.

126. Berditchevski F, Tolias K F, Wong K, Carpenter C L, Hemler ME. A novel link between integrins, transmembrane –4 superfamilyproteins (CD63 and CD81), and phosphatidylinositol 4-kinase. J

Biol Chem 1997; 272: 2595–98.127. Sinigaglia F, Torti M, Ramaschi G, Balduini C. Fibronectin

modulates the activation of human platelets. Biochem Biophys

Res Commun 1989; 165: 966–72.

128. Nadal F, Levy-Toledano S, Grelac F, Caen J P, Rosa J P, BryckaertM. Negative regulation of mitogen-activated protein kinaseactivation by integrin aIIbb3 in platelets. J Biol Chem 1997; 272:

22381– 84.129. Nurden P, Poujol C, Winckler J, Combrie J, Nurden A T, Caen J

P. Interaction of the granular pool of fibrinogen with GPIIb–IIIa

in the platelets of a Glanzmann thrombastenia variant with a752Ser®Pro mutation in GPIIIA. Thromb Haemost 2000; 82(Suppl): 499.

130. Wang R G, Shattil S J, Ambruso D R, Newman P J. Truncation ofthe cytoplasmic domain of b3 in a variant form of Glanzmannthrombasthenia abrogates signaling through the integrin aIIbb3

complex. J Clin Invest 1997; 100: 2393–403.131. Lee S B, Rao A K, Lee K H, Yang X, Bae Y S, Rhee S G.

Decreased expression of phospholipase C-b2 isozyme in humanplatelets with impaired function. Blood 1996; 88: 1684– 91.

132. Gabbeta J, Yang X, Kowalska M A, Sun L, Dhanasekaran N,Rao A K. Platelet signal transduction defect with Ga subunitdysfunction and diminished Gaq in a patient with abnormal

platelet responses. Proc Natl Acad Sci USA 1997; 94:8750– 55.

133. Feng D, Lindpaintner K, Larson M G, Rao V S, O’Donnell C J,

Lipinska I et al. Increased platelet aggregability associated withplatelet GPIIIa PlA2 polymorphism: the Framingham OffspringStudy. Arterioscler Thromb Vasc Biol 1999; 19: 1142–47.

134. Vijayan K V, Goldschmidt-Clermont P J, Roos C, Bray P F. ThePlA2 polymorphism of integrin b3 enhances outside-in signalingand adhesive functions. J Clin Invest 2000; 105: 793– 802.

PLATELETS 319

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Chapter III

Involvement of the β3 E749

ATSTFTN756

region in stabilizing the

αIIbβ3-ligand interaction

Pieter E.M.H. Litjens, Gertie Gorter, Jari Ylänne, Jan-Willem N. Akkerman and Gijsbert van Willigen

Journal of Thrombosis and Haemostasis, 2003, 1, 2216-2224

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Page 50: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

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-58-

Page 59: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

Chapter IV

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in

platelet adhesion on fibrinogen under flow conditions

Pieter E.M.H. Litjens, Christine I. Kroner, Jan-Willem N. Akkerman and Gijsbert van Willigen

Journal of Thrombosis and Haemostasis, 2003, 1, 2014-2021

-59-

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-67-

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Chapter V

A tripeptide mimetic of von Willebrand factor residues 981–983

enhances platelet adhesion to fibrinogen by signaling through

integrin αIIbβ3

Pieter E . M. H. Litjens, Gijsbert van Willigen, Cees Weeterings, Martin J . W.

IJsseldijk, Marjolein van Lier, Erkki Koivunen, Carl. G. Gahmberg and Jan-Willem

N. Akkerman

Journal of Thrombosis and Haemostasis, 2005, 3: 1274–83.

-68-

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ORIGINAL ARTICLE

A tripeptide mimetic of von Willebrand factor residues 981–983enhances platelet adhesion to fibrinogen by signaling throughintegrin aIIbb3

P . E . M. H . L I T J ENS ,* G . VAN WILL IGEN ,* C . WEETER INGS ,* M. J . W. I J SSELD I JK ,* M. VAN L IER ,*

E . KOIVUNEN,� C. G . GAHMBERG� and J . W . N . AKKERMAN**Laboratory for Thrombosis and Haemostasis, Department of Haematology, UMCU, and Institute for Biomembranes, Utrecht University, Utrecht,

The Netherlands; and �Division of Biochemistry, Faculty of Biosciences, University of Helsinki, Helsinki, Finland

To cite this article: Litjens PEMH, van Willigen G, Weeterings C, Ijsseldijk MJW, van Lier M, Koivunen E, Gahmberg CG, Akkerman JWN.

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin aIIbb3.

J Thromb Haemost 2005; 3: 1274–83.

Summary. Background: RGD is a major recognition sequence

for ligands of plateletaIIbb3. Objective andmethods: To identify

potential binding sites for aIIbb3 apart from RGD, we screened

phage display libraries by blocking the enrichment of RGD-

containing phages with a GRGDS peptide and identified a

novel integrin recognition tripeptide sequence, VPW. Results:

Platelets adhered to an immobilized cyclic VPW containing

peptide in a aIIbb3-dependent manner; platelets and aIIbb3-

expressing CHO cells adhered faster to immobilized aIIbb3-

ligands in the presence of soluble VPW. In platelets adhering to

fibrinogen, VPW accelerated the activation of the tyrosine

kinase Syk which controls cytoskeletal rearrangements. In

aIIbb3-expressing CHO cells, VPW induced a faster formation

of stress fibers. Sequence alignment positioned VPW to V980–

P981-W982 in the vonWillebrand factor (vWf) A-3 domain. In

blood from a vWf-deficient individual, VPW increased platelet

adhesion to fibrinogen but not to collagen under flow

and rescued the impaired adhesion to vWf deficient in

A-3. Conclusion: These data reveal a VPW sequence that

contributes to aIIbb3 activation in in vitro experiments.Whether

theV980–P981-W982 sequence in vWf shows similar properties

under in vivo conditions remains to be established.

Keywords: adhesion, glycoprotein IIb-IIIa, integrin aIIbb3,

phage display, Syk, von Willebrand factor.

Introduction

The integrin aIIbb3 (glycoprotein IIb/IIIa or CD41b/CD61)

plays an essential role in hemostasis and thrombosis as it

mediates platelet–platelet bridging through binding of soluble

and adhesion to immobilized adhesive proteins. In suspension,

binding of fibrinogen, von Willebrand factor (vWf) and

fibronectin requires stimulation by platelet activating agents

such as thrombin or ADPwhich through intracellular signaling

shift the integrin from a low to a high affinity state triggering

ligand binding. At adhesive surfaces, platelets bind aIIbb3ligands without prior stimulation illustrating that low affinity

aIIbb3 is responsive for binding to immobilized ligands. The

platelet inhibitor prostacyclin converts aIIbb3 to an inaccessible

conformation and completely abolishes aIIbb3-dependent

adhesion. Following ligand binding, the integrin forms clusters

leading to an increase in avidity and binds to cytoskeletal

proteins such as talin, an important mediator of adhesion. The

combined affinity and avidity changes facilitate integrin–ligand

interaction and initiate signaling pathways that form stress

fibers and focal adhesions [1,2].

Interestingly, certain treatments make ligand binding sites

accessible via extracellular interaction with the integrin. The

RGD motif is present in many integrin ligands and RGD

peptides and mimetics bind to aIIbb3 thereby interfering with

platelet adhesion and aggregation [3]. Frelinger et al. [4]

reported an antibody that recognizes epitopes on RGD-

occupied aIIbb3. One of these ligand-induced binding site

(LIBS)-antibodies called LIBS6 induced binding of soluble

ligands. Derrick et al. [5] designed the peptide LSARLAF, that

was supposed to inhibit binding of soluble fibrinogen but

enhanced platelet adhesion to fibrinogen. Stimulation by

LSARLAF was independent of intracellular mechanisms and

not affected by cAMP, which is a potent inhibitor of aIIbb3activation by physiological stimulators. The b2-integrin LFA-1

was activated byGSLEVNCSTTCNQPEVGGLETSY, which

is derived from the first Ig-domain of ICAM-2, illustrating that

peptide activation of integrins is not restricted to aIIbb3 [6].

Correspondence: Jan Willem N. Akkerman, Thrombosis and

Haemostasis Laboratory, Department of Haematology (G03.647),

University Medical Center Utrecht, PO Box 85.500, 3508 GA Utrecht,

The Netherlands.

Tel.: +31 30 250 6512; fax: +31 30 251 1893; e-mail: j.w.n.akkerman@

lab.azu.nl

Received 25 October 2004, accepted 16 March 2005

Journal of Thrombosis and Haemostasis, 3: 1274–1283

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Binding to the I-domain of the b2-chain changed the affinity of

the integrin, facilitating binding of different ICAMs. The

activation was independent of intracellular signal generation,

but required metabolic energy and an intact cytoskeleton.

Platelet aIIbb3 binds fibronectin and vWf through their RGD

sequence and the peptide GRGDS is an efficient blocker of

ligand binding. To identify possible secondary recognition sites

for aIIbb3, we screened phage display libraries by blocking the

enrichment of RGD-containing phages with a GRGDS

peptide and identified a novel integrin recognition tripeptide

sequence, which is present in vWf, a major component in the

attachment of platelets to the damaged vessel wall.

Materials and methods

Reagents

We used phalloidin-tetramethylrhodamine isothiocyanate

(TRITC), indomethacin, cytochalasin D, p-nitrophenylphos-

phate tetracycline, kanamycin and human collagen type III

(Sigma, St Louis, MO, USA), purified aIIbb3 integrin (Enzyme

Research Laboratories, Swansea, UK), fibrinogen (FIB3;

plasminogen-, vWf- and fibronectin-free; Enzyme Research

Laboratories, Lafayette, IN, USA), GRGDS-peptide and

fibrinogen c-peptide HHLGGAKQAGDV (Bachem, Buben-

dorf, Switzerland), the antibodies anti-aIIbb3 P2 (Coulter-

Immunotech,Marseille, France), anti-avb3 LM609 (Chemicon,

Temecula, CA, USA), anti-b2 7E4 [7], anti-b1 mAb 13 (a kind

gift of Dr K. Yamada, NIH, Bethesda, MD, USA), anti-Syk

4D10and anti-SykLR (SantaCruzBiotechnology, SantaCruz,

CA, USA), anti-phosphotyrosine antibody 4G10 (Upstate

Biotechnology, Lake Placid, NY, USA); anti-b3-subunit 4A7,

GAMPO, goat-anti-mouse FITC-labeled and GAM-FITC

(DAKO, Glostrup, Denmark). Synthetic cyclic peptides CIV-

PWGRYC (VPW), cyclic CIVHWGRYC (VHW) and cyclic

CGYLPLRYVC (YLP-peptide) were synthesized as described

or fromGenosphere Biotech (Paris, France). Inhibitors (15 min

preincubation, 22 �C) were indomethacin (30 lM; Sigma, St

Louis,MO,USA), PP1 (5 lM), piceatannol (10 lM), SB 203580

(10 lM; Alexis, San Diego, CA, USA); PD98059 (10 lM;

Calbiochem Corporation, La Jolla, CA, USA) and bis-

indolylmaleimide I (5 lM; Roche, Basel, Switzerland). Protein

A linked sepharose CL-4B and protein A horseradish peroxi-

dase-labeled (proteinAHRP)were fromAmershamPharmacia

Biotech (Uppsala, Sweden) andEnhancedChemoluminescence

from NEN (Perkin Elmer Life Sciences, Brussels, Belgium).

Recombinant vWfandDA-3vWfwas preparedas described [8].

Peptide synthesis and phage display

Peptides were synthesized by Fmoc-chemistry on an Applied

Biosystems model 433A (Foster City, CA, USA). Disulphide

bonds were formed by oxidation in 10 mmol L)1 ammonium

bicarbonate buffer (pH 9.0) overnight at 22 �C. Peptides were

purified by HPLC; generation of disulphide bonds was

confirmed by mass spectrometry. Peptides were >98% pure

based on HPLC. aIIbb3 preparations were diluted in Tris-

buffered saline (TBS, pH 7.5) containing 25 mmol L)1 n-octyl-

b-D-glucopyranoside and 1 lg per well was coated onto

microtiter plates (overnight, 4 �C). Wells were blocked with

5% BSA-TBS (1 h, 22 �C) and washed with TBS. Biopanning

was based on a CX7C phage display library [9] in the presence

of 500 lmol L)1 GRGDS. Phage binding, elution and ampli-

fication in E. coli K91kan cells were repeated five times. After

the fourth and fifth panning, bacterial colonies were collected

and stored in 10 lL of TBS at )20 �C. For direct colony

sequencing [10], 1 lL samples were subjected to PCR with

10 pmol of forward (5¢-TAATACGACTCACTATAGGGC

AAGCTGATAAACCGATACAATT-3¢) and reverse primer

(5¢-CCCTCATAGTTAGCGT AACGATCT-3¢). PCR condi-

tions were 92 �C, 30 s; 60 �C, 30 s and 72 �C, 60 s; 35 cycles.

One microliter of PCR reaction was taken for sequencing with

15 pmol primer and analyzed on an ABI 310 (PE Applied

Biosystems, Foster City, CA, USA). Multiplication was in

E. coli K91kan cells grown with kanamycin and tetracycline.

Phages were precipitated with polyethylene glycol/NaCl,

resuspended in TBS and stored at 4 �C until use.

Preparation of platelets and CHO cells expressing aIIbb3

Venous blood was collected from healthy donors and from a

patient with type III von Willebrand disease (<1% plasma-

and platelet vWf, 11) into 1/10 vol of 130 mmol L)1 tri-sodium

citrate. The donors had not taken any medication during the

past 10 days.Washed platelets were prepared by centrifugation

(10 min, 150 g, 22 �C), acidification to pH 6.5 and a second

centrifugation (15 min, 330 g, 22 �C) and resuspension in

HEPES-Tyrode (145 mmol L)1 NaCl, 5 mmol L)1 KCl,

0.5 mmol L)1 Na2HPO4, 1 mmol L)1 MgSO4, 10 mmol L)1

HEPES, pH 7.2) containing 0.2% BSA and 5.6 mmol L)1

D-glucose. Wild type Chinese hamster ovary cells (CHO-wt)

and cells stably expressing the integrin aIIbb3 (A-3 cells, CHO-

aIIbb3), a gift of Dr J. Ylanne, Helsinki University, Finland

were cultured as described [12].

Static adhesion of platelets and CHO cells

Microtiter wells were coated overnight (4 �C) with 50 lL of

100 lg mL)1 VPW, GRGDS, fibrinogen c-peptide

(HHLGGAKQAGDV), collagen or 10 lg mL)1 vWf or

fibronectin. Adhesion was studied in the presence of GRGDS

and c-peptide, EDTA (5 mM) and anti-aIIbb3, -avb3, -b2 and -b1antibodies (20 lg mL)1). In experiments with soluble VPW,

VHW and the YLP-control, peptides were present at

100 lg mL)1; the vehicle was 0.1% DMSO. After coating,

wells were blocked with 5% BSA in TBS (1 h, 22 �C) and

washed three times with HEPES-Tyrode (0.2% BSA, 0.1%

D-glucose). Adhesion was started by adding 100 lL platelets

(150 000 lL)1) or CHO cells (50 000 per well). Wells were

incubated (60 min, 37 �C), washed five times with HEPES-

Tyrode (pH 7.2, 0.2% BSA, 0.1% D-glucose) and adhered

platelets detected by cellular phosphatase assay [13].

Integrin aIIbb3 activation by VPW peptide 1275

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Confocal microscopy

Glass coverslips were coated overnight (4 �C) with 50 lL

fibrinogen (10 lg mL)1), blocked with 5% BSA in TBS

(1 h, 22 �C) and washed three times with HEPES-Tyrode

(pH 7.2. 0.2% BSA, 0.1% D-glucose). Cells were pretreated

with VPW, VHW and the control YLP-peptide

(100 lmol L)1) for 30 min, 22 �C. Following addition of

50 000 CHO-cells, adhesion was measured after 60 and

90 min (37 �C) after washing with HEPES-Tyrode and

fixation with 200 lL of 4.5% paraformaldehyde in PBS,

0.1% Tween-20 (5 min; 22 �C). Cells were incubated in

0.1 mol L)1 ammonium chloride and twice incubated in

0.013 mol L)1 borohydride in PBS (5 min; 22 �C). Cover-

slips were washed, incubated with 165 nmol L)1 phalloidin-

TRITC (in PBS; 30 min; 22 �C) followed by GAM-FITC,

washed in PBS, embedded in Mowiol and analyzed by

confocal microscopy.

Syk tyrosine phosphorylation

Samples were prepared from non-adhered and adhered plate-

lets by addition of 100 lL 2· ice-cold lysis buffer (1 mmol L)1

orthovanadate, 1 lg mL)1 leupeptin, 1 mmol L)1 PMSF, 1%

v/vNP-40, 0.5%w/v deoxycholate and 0.1%w/v SDS in PBS).

Fifty microliters of 10% protein A Sepharose and 1 lg mL)1

anti-Syk antibody was added and incubated (3 h, 4 �C).

Precipitates were washed and 30 lL 3· sample buffer was

added. Samples were boiled (5 min), proteins separated by

SDS-PAGE (8%) and blotted with appropriate antibody.

Bands were visualized by proteinA-HRP-labeled andECL and

quantified (ImageQuant; Amersham Biosciences, Uppsala,

Sweden).

Perfusion experiments

Coverslips were coated with 100 lg mL)1 fibrinogen or

10 lg mL)1 vWf or vWf DA-3 for 1 h at 22 �C or sprayed

with collagen (30 lg cm)2) and blocked with human serum

albumin. Whole blood in 0.1 volume 130 mM trisodium citrate

containing VPW, VHW and YLP control (100 lg mL)1) was

perfused in a single passage parallel plate perfusion chamber

[14]. Coverslips were collected, rinsed in HEPES-buffered

saline (10 mmol L)1 HEPES, 0.15 mmol L)1 NaCl, pH 7.4),

fixed in 0.5% glutaraldehyde in PBS, dehydrated in methanol

and stained with May-Grunwald-Giemsa. Platelet adhesion

was evaluated with a light microscope equipped with a

JAI-CCD camera coupled to a Matrox frame grabber using

Optimas 6.2 (Optimas Inc., Seattle, WA, USA) software for

image analysis and expressed as percent surface coverage.

Statistics

Data are expressed as mean ± SD (n ¼ 3–6) and analyzed by

Student’s t-test for paired observations. Differences were

considered significant at P < 0.05.

Results

Identification of an integrin aIIbb3-binding VPW motif

ACX7C phage display library was used to search for peptides

that bind to integrin aIIbb3 in the presence of GRGDS. After

the fifth round of phage selection, a 1000-fold enrichment was

obtained on the coated integrin compared with coated BSA.

Sequencing of the bound phage revealed only four RGD-

containing sequences (CWARGDFRC, CEPRGDWRC,

CVARGDWRC, CWARGDPRC). Consistent with our

selection strategy the majority of sequences lacked RGD

and revealed novel binders. The consensus in these sequences

was the tri-amino acid motif Val-Pro-Trp (VPW; Fig. 1). All

sequences contained a positively charged arginine residue,

showing a VPWXR consensus. The most strongly enriched

sequence was CIVPWGRYC, which was observed six times.

Two peptides also contained the conservative Val to Ile

substitution yielding the IPW motif. Data base search

indicated that a VPW motif with a IDVPWNVVP sequence

CIVPWGRYC (6) CKVPWARWC

CLVPWGRLC

(1)

(1)

CNVPWGRYC (1)

CDVPWRDLC (1)

CVPWRDWTC (1)

CLIPWGRFC (4)

CIPWGRYFC (1)

(3)

(3)

(3)

(2)

(1)

(1)

CLVPWGRYC

CAVPWARYC

CAVPWGRLC

CLVPWARWC

CAVPWGRYC

CIVPWARYC

von Willebrand Factor: I(978)DVPWNVVP

100

75

ph

ag

e b

ind

ing

phage peptide

(% o

f m

axim

al b

ind

ing

)

50

25

0CIVPWGRYC CLVPWGRYC CAVPWARYC CVARGDWRC

Fig. 1. Binding of VPW- and RGD-containing phage to immobilized

aIIbb3. Top: Phage sequences that bound to aIIbb3 and alignment with

A3-domain of von Willebrand factor (consensus binding motif in bold;

number of sequenced clones in parenthesis). Bottom: Microtiter wells

coated with integrin aIIbb3 incubated with phage expressing indicated

sequences (1 h, 22 �C) without (open bars) and with (hatched bars)

500 lmol L)1 GRGDS.

1276 P. E. M. H. Litjens et al

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is present on the A-3 domain of vWf, the major aIIbb3 ligand.

Phage attachment assays were carried out with the VPW-

displaying phages and RGD-displaying phage to compare

their binding specificity to aIIbb3. Each VPW-displaying

clone bound to aIIbb3 with a similar efficiency as the

CVARGDWRC phage, one of the RGD-displaying phages

discovered by panning. GRGDS peptide showed little or only

partial competition with VPW-phage binding, whereas bind-

ing of the CVARGDWRC phage was almost completely

inhibited.As thephagecontainingCIVPWGRYCshowed the

strongest binding activity, we synthesized the corresponding

cyclic peptide. The peptide blocked binding of VPW-contain-

ingphages butnotof theRGD-containingphage (not shown).

Adhesion of platelets to surface-coated VPW and ligands

of aIIbb3

VPW neither induced aggregation or secretion in stirred

platelet suspensions nor changed these responses induced by

platelet agonists (not shown). To investigate whether the

peptide affected adhesion, VPW was coated onto microtiter

plates. Platelets adhered to immobilized VPW but not to

GRGDS, fibrinogen c-peptide or BSA. Thus, adhesion

appeared specific for VPW (Fig. 2A). To investigate the role

of aIIbb3, platelets were pretreated with aIIbb3 antagonists

GRGDS, fibrinogen c-peptide or EDTA. These treatments

inhibited adhesion to VPW, albeit to different extents

(Fig. 2B). Similar studies with anti-integrin antibodies showed

70% inhibition by aIIbb3 antibody P2, but antibodies against

avb3, b1- and b2-integrins had no effect (Fig. 2C). Titration of

VPW revealed a dose-dependent adhesion (Fig. 2D). These

data indicate that platelets adhere to VPW via integrin aIIbb3.

In the presence of VPW there was a three- to fourfold higher

adhesion to fibrinogen, vWf and fibronectin but not to collagen

(Fig. 3A). Microscopic evaluation showed increased coverage

of a fibrinogen-coated surface in the presence of VPW

(Fig. 3B,C). Thus, VPW enhanced platelet adhesion to

surface-bound ligands of aIIbb3.

2.0A B

C D

1.5

1.0

Ab

s.

40

5 n

m

Ab

s.

40

5 n

m

0.5

0.0

2.0

1.5

1.0

Abs.

40

5 n

m

Abs.

40

5 n

m

0.5

0.0

2.0

1.5

1.0

0.5

0.0

0.01 0.1

coated VPW peptide (µg/well)

1 10

1.5

1.0

0.5

0.0VPW

contr

ol

αllb

β3 (

P2)

β2 (

7E

4)

β1 (

mA

B 1

3)

αvβ3 (

LM

609)

GRGDS

coated peptide

coated VPW

coated VPW

BSA controlγ-peptide GRGDS EDTAγ-peptide

Fig. 2. Static adhesion of platelets to immobilized VPW. (A)Wells coated as indicated incubated with platelets (1 h, 37 �C) and adhesionmeasured by acid

phosphatase assay. (B) VPW-coated wells incubated with platelets pretreated with GRGDS, c-peptide or EDTA (30 min, 22 �C). (C) VPW-coated wells

incubated with platelets pretreated with antibodies against indicated integrins (30 min, 22 �C). (D) Dose–response curve of platelet adhesion to coated

VPW. Results are expressed as absorbance at 405 nm. Apart from data from avb3-, b1- and b2 antibodies, differences were significant.

Integrin aIIbb3 activation by VPW peptide 1277

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VPW enhances platelet adhesion to fibrinogen via

intracellular signaling

Stimulation of adhesion by VPW suggested that VPW induced

a conformational change in aIIbb3 which enhanced ligand

binding to the integrin and outside-in signaling to cytoskeletal

re-arrangements.When platelets were incubatedwith inhibitors

of signaling pathways there was little change in adhesion to

coated VPW (not shown). When platelet suspensions were first

incubatedwith these inhibitors and thereafter allowed to adhere

to fibrinogen, different results were obtained. Again, inhibition

of COX1 had no effect but inhibition of Src kinases and

especially inhibition of Syk strongly interfered with stimulation

by VPW, suggesting that VPW supported adhesion through

Src-family members and Syk (Fig. 4A). However, prolonged

incubation of platelet suspensions with VPW in concentrations

up to 200 lg mL)1 failed to induce Syk phosphorylation

(Fig. 4B). In contrast, when Syk analysis was restricted to

platelets adhered to fibrinogen, therewas a threefold higher Syk

activation in the presence of VPWcompared with sham-treated

cells. Also when Syk phosphorylation was expressed as a ratio

over total Syk to account for different numbers of adhered cells,

stimulation byVPWwas evident.WithGRGDS therewas little

adhesion and no Syk activation could be detected (Fig. 4C–E).

Also a circular peptide with a P-H substitution (VHW), which

was designed to evaluate the role of the P residue, increased Syk

phosphorylation but much weaker than VPW. In contrast, a

peptide with a random replacement of neutral amino acids

(YLP-peptide) had no effect (not shown).

VPW enhances adhesion of aIIbb3-expressing CHO cells

To understand involvement of aIIbb3 in stimulation by

VPW, CHO-aIIbb3 cells were adhered to coated VPW and

2.0A

B C

1.5

1.0

0.5

0.0fibrinogen

control VPW

coated fibrinogen

fibronectin collagenvWf

coated protein

Ab

s.

40

5 n

m

Fig. 3. Effect of VPW on platelet adhesion. (A) Wells coated as indicated and incubated with platelets preincubated with VPW (hatched bars) or vehicle

(open bars; 5 min, 22 �C). Differences were significant. Microscopic evaluation of static platelet adhesion to fibrinogen without (B) and with (C)

preincubation with VPW.

1278 P. E. M. H. Litjens et al

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static adhesion was analyzed by the cellular phosphatase

assay. There was a basal adhesion of CHO-wt that was

unaffected by GRGDS and therefore independent of aIIbb3.

With CHO-aIIbb3 adhesion to VPW increased and RGDS

reduced this effect to the range found with wt-CHO

(Fig. 5A). There was little adhesion by CHO-wt to fibrin-

ogen ± VPW (Fig. 5B), VHW and vehicle (not shown). In

contrast, there was a fourfold higher adhesion of CHO-

aIIbb3 which increased further with about 70% in the

presence of VPW. Adhesion of CHO-aIIbb3 to fibrinogen

was strongly reduced by GRGDS (Fig. 5B). Morphometric

analysis showed a much faster shift of CHO-aIIbb3 from the

round to the spread morphology induced by VPW and to a

lesser extent by VHW compared with control YLP peptide

750A

C

E

D

B

500

250

% o

f co

ntr

ol

Syk t

ota

l

Syk-P

/Syk t

ota

l

0

none

5 3

2

1

0

4

3

2

1

0vehicle VHW VPW

vehicle VPW VHW VPW VHW

Syk -P

Syk -T

GRGDS

vehicle VHW VPW

indo PP1 piceatannol vehicle VPW VHW

Syk -T

Syk -P

Fig. 4. Activation of Syk by VPW in platelets adhered to fibrinogen. (A) Effect of metabolic inhibitors on platelet adhesion to fibrinogen without (white

bars) and with (hatched bars) VPW. Data are percent of controls (without VPW and inhibitors). Differences were significant. (B) Platelet suspensions

incubated with vehicle, VPW and VHW. After 120 min, cells were analyzed for tyrosine phosphorylated (Syk-P) and total Syk (Syk-T). (C–E) Platelets

treated with vehicle VPW, VHW±GRGDS and incubated in wells coatedwith fibrinogen. After 120 min, adhered cells were analyzed for Syk-T and Syk-

P/Syk-T (to account for different adhesion caused by peptides). Control peptide YLP (200 lM, 120 min, 37 �C) gave the same results as vehicle (not

shown). Differences between VHW- and VPW-treated platelets were different.

Integrin aIIbb3 activation by VPW peptide 1279

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(Fig. 5C,D). Confocal images showed that the faster

spreading was accompanied by a faster formation of stress

fibers (Fig. 5E). Together these data show that VPW, and

to a lesser extent VHW, enhance the adhesion of CHO cells

in a aIIbb3-dependent manner through cytoskeletal

re-arrangements.

150A B

C

E

D

100

% o

f m

axim

al b

ind

ing

% o

f m

axim

al b

ind

ing

GRGDS

CHO-wt CHO-wt

coated VPW coated fibrinogen

CHO-α llbβ3 CHO-α llbβ3

GRGDS

GRGDS

50

0

70

60

50

40

30

cell

num

be

r (%

of

tota

l)

20

10

0

70YLP

VHP

VPW

60

50

40

30

20

10

0

round intermediate

coated fibringen

YLP VPW VHW

60 min

90 min

10 µm

10 µm

10 µm

10 µm

10 µm

10 µm

coated fibringen

60 minutes 90 minutes

spread round intermediate spread

150

100

50

0

Fig. 5. Effect of VPW on static adhesion of aIIbb3 expressing CHO cells. (A) Adhesion of wild type and CHO-aIIbb3 to coated VPW ± GRGDS. (B)

Adhesion of CHO cells to fibrinogen following pretreatment with vehicle (open bars) and VPW (hatched bars) ± GRGDS. VPW induced a significant

difference in adhesion of CHO-aIIbb3. (C, D) Morphology changes of CHO-aIIbb3 during adhesion to fibrinogen based on confocal images (expressed as

percent of total cell number). Morphological criteria were based on (E): round cells as shown for YLP control; intermediate and spread as shown for cells

treated with VPW for 60 and 90 min respectively. Differences between the three types of round and spread cells were significant. (E) Confocal images

stained for actin of CHO-aIIbb3 adhered to fibrinogen in the presence of YLP, VHW and VPW.

1280 P. E. M. H. Litjens et al

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Platelet adhesion under flow

To study the effect of flow, adhesion was measured in a

perfusion chamber. In normal blood VPW did not change

adhesion to coated rec-vWf, fibrinogen or collagen. Also in

washed platelets added to red blood cells in buffer VPW failed

to change surface coverage (not shown). In experiments with

blood from a patient with a severe vWf deficiency VPW

induced a threefold increase in adhesion to fibrinogen, reaching

the range found in the presence of vWf. An almost similar

increase was observed with VHW whereas the YLP control

had no effect. In contrast, adhesion to collagen was unchanged

(Fig. 6A,C,D). VPW is present in the vWfA-3 domain which is

known for its binding properties to collagen. Blood from the

vWf-deficient patient showed a lower adhesion to vWf DA-3

than to wild type vWf amounting to a decrease of about 70%

at 300 s)1 to 30% at 1600 s)1 (Fig. 6B). The presence of VPW

led to a partial (at 300 s)1) to complete (at 1600 s)1) recovery,

an effect also seen with VHW. These data indicate that VPW

and VHW rescue impaired adhesion to a vWf construct

deficient in the VPW-containing A-3 domain.

Discussion

A novel cyclic peptide, CIVPWGRYC (VPW) enhances

interaction between platelet aIIbb3 and immobilized ligands.

Phage library screenings have previously identified the triamino

acid motifs RGD, NGR, LDV and LLG as minimal recog-

nition sequences for integrins [15]. So far, phage display studies

with aIIbb3 resulted mainly in RGD and KGD containing

500A B

C D

150300 s–1 1600 s–1

100

50

400

300

200

rela

tive p

late

let adhesio

n (

%)

rela

tive p

late

let adhesio

n (

%)

100

0 0none VHW VPW

∆ A-3 vWF

none VPWYLP VHW VHWVPW

fibrinogen

fibrinogen

control VPW

collagen

VPWvWF

Fig. 6. Effect of VPWon platelet adhesion under flow. (A) Platelet adhesion to fibrinogen and collagen after 3 min perfusion at 1600 s)1 (37 �C) of citrated

whole blood from a patient with type III von Willebrand disease. Immediately before start of the perfusion, 100 lg mL)1 control YLP-peptide, VHW,

VPW and 15 lg mL)1 rec-vWf were added. Adhesion with peptides was expressed as percent of suspensions without additions. (B) Adhesion of patient

blood over a surface coated with DA-3 vWf. Adhesion to DA-3 vWf without and with peptides was expressed as percent of adhesion of von Willebrand

Disease type III platelets to rec-vWf without additions. Platelet count: 2.4–3.3 · 1011, MPV 6–7 · 10)15 L, Ht: 30. Adhesion to fibrinogen and DA-3 vWf

between YLP/none and other treatments were significant. Platelet adhesion to fibrinogen without (C) and with (D) VPW.

Integrin aIIbb3 activation by VPW peptide 1281

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binding peptides and the strong enrichment of these motifs

during panning prevented identification of other interacting

sequences.

The binding site of VPW on the integrin must be close to the

RGD-recognition site. Although GRGDS peptide hardly

interfered with binding of VPW-phage to aIIbb3, it competed

effectively with platelet adhesion to VPW. In this sense

CIVPWGRYC resembles the peptide CRRETAWAC. The

specific binding of CRRETAWAC to integrin a5b1 is inhibited

by RGD [9], although antibody mapping shows that CRRE-

TAWAC and RGD recognize different areas of the integrin

[16]. One benefit of integrin ligand peptides developed by phage

display is that theymay help to define the borders of the ligand-

binding pocket of an integrin. We anticipate that VPW,

together with antibodies that recognize the active integrin,

could be useful to determine how aIIbb3 is converted to a fully

active state.

Platelets adhered readily to immobilized VPW but not to

other aIIbb3-binding peptides and an antibody against aIIbb3and aIIbb3-blocking peptides interfered with binding. This

indicates that immobilized VPW binds to aIIbb3 with a high

affinity. It is therefore conceivable that VPW-aIIbb3 interaction

initiates platelet functions. However, VPW addition to platelet

suspensions failed to induce aggregation/secretion or to prime

platelets to agonist stimulation. VPW also failed to activate

Syk. In contrast, VPW strongly increased adhesion to immo-

bilized fibrinogen, vWf and fibronectin measured under static

conditions. Apparently, VPW interfered with aIIbb3 through a

mechanism that increased ligand binding irrespective of the

type of ligand. A similar stimulation of adhesion to fibrinogen

was observed with aIIbb3 expressing CHO cells. VPW did not

interfere with adhesion to collagen.

To investigate the effect of VPW and VHW on platelet

adhesion under flow, peptides were added before the start of

the perfusions and adhesion to fibrinogen, rec-vWf and

collagen were investigated. In normal blood neither VPW nor

VHW changed platelet adhesion to surface-bound aIIbb3ligands. Also suspensions of erythrocytes and platelets

in buffer failed to show VPW-dependent adhesion. However,

in vWf-deficient blood VPW induced a threefold increase in

adhesion to fibrinogen. Also VHW enhanced adhesion under

flow and induced more stimulation than seen under static

conditions. The control YLP peptide had no effect. Adhesion

in vWf-deficient blood to coated vWf DA-3 was lower than to

intact vWf illustrating a role of the A-3 domain in platelet

adhesion to vWf. In the presence of VPW and VHW adhesion

increased to the range found with intact vWF demonstrating

that the peptides could compensate the loss of VPW in the A-3

domain. Neither VPW nor VHW changed platelet adhesion to

collagen as observed under static conditions. Under high shear,

platelet adhesion to the injured vessel wall is initiated by the

binding of glycoprotein Ib to the A-1 domain of vWf. This

interaction supports tethering and rolling and is followed by a

second phase supported by aIIbb3 binding to the C-1 domain of

vWf which promotes platelet arrest [17]. VWf binding to

glycoprotein Ib starts a signaling cascade involving changes in

cytosolic Ca2+ and recruitment of Src family kinases leading to

cytoskeletal re-arrangements that suppert firm adhesion. In

addition, glycoprotein Ib mediated signaling activates aIIbb3enabling further vWf binding through its C1 domain initiating

specific Ca2+ fluxes and changes in protein kinase C activity

[18].

Adhesion to fibrinogen in the presence of VPWwas inhibited

by the Src kinase inhibitor PP1 and the Syk inhibitor

piceatannol. Immunoprecipitation confirmed that VPW

enhances Syk phosphorylation in adhering platelets. The

partial inhibition by PP1 agrees with the fact that Src is an

upstream kinase of Syk. The peptide with a P fi H substitu-

tion (VHW) had a much smaller effect. Recent studies show

that aIIbb3 is constitutively associated with the tyrosine kinases

Src and Csk. Adhesion to fibrinogen triggers the release of Csk,

which is a negative regulator of Src, making possible the

tyrosine phosphorylation of aIIbb3-bound Src. This step

triggers the recruitment of Syk to aIIbb3 resulting in its

activation and further signaling to effectors that control

cytoskeletal reassembly such as Vav1 and SLP-76 [19]. The

present findings are in agreement with this concept. VPW

stimulated the phosphorylation of Syk in adhering platelets but

not in platelets in suspension which are unable to bind soluble

fibrinogen. Hence, ligand binding to aIIbb3 is a prerequisite for

VPW signaling to Syk.

In the crystal structure of vWf, the VPW sequence is buried

between the b3-sheet and the a2-helix of the A3 domain and is

quite far from the major integrin-binding site, the RGD

sequence in the COOH terminus [8,20]. This position would

argue strongly against a role of this domain in vWf function.

Two recent reports indicate that this site might be accessible to

other proteins. First, using phage display, mutant analysis and

peptide modeling, Vanhoorelbeke et al. [21] found an anti-

human vWfMoAb (82D6A3) against the vWfA3-domain that

bound to the sequence SPWR which could be aligned to P981-

W982 in the A3 domain of vWf. The antibody inhibited vWf

binding to fibrillar collagens types 1 and III in vitro and

interfered with arterial thrombus formation in baboons [22].

Hence, this domain is accessible in vivo. Secondly, by fluores-

cence anisotrophy analysis of the W982 residue in vWf A3

domain, Hellings et al. [23] detected two different rotamers, one

that corresponds with the position of W983 in the X-ray

structure and one with properties of an exposed rotamer

conformation. It is therefore attractive to speculate that the

VPW region in vWf changes its conformation upon surface

binding making the hydrophilic conformation of the VPW

region more accessible to platelet integrin aIIbb3. Definite proof

for such a role under in vivo conditions awaits further

investigation.

Acknowledgements

The cooperation of the patient is gratefully acknowledged. The

authors thank Drs H.M. van den Berg and H.F. Heijnen for

support. This work was supported by the Netherlands

Organization for Scientific Research (GR91-266, 902-26-193),

1282 P. E. M. H. Litjens et al

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the Academy of Finland, the Sigrid Juselius Foundation and

the Finnish Cancer Society. GvW is a research fellow of the

Catharijne Foundation and supported by the Dirk-Zwager

Assink Foundation. JWNA is supported by the Netherlands

Thrombosis Foundation.

References

1 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet

paradigm. Blood 1998; 91: 2645–57.

2 Beumer S, IJsseldijk MJW, De Groot PG, Sixma JJ. Platelet adhesion

to fibronectin in flow: dependence on surface concentration and shear

rate, role of platelet membrane glycoproteins GP IIb/IIIa and VLA-5,

and inhibition by heparin. Blood 1994; 84: 3724–33.

3 D’Souza SE, Ginsberg MH, Plow EF. Arginyl-glycyl-aspartic acid

(RGD): a cell adhesion motif. Trends Biochem Sci 1991; 16: 246–50.

4 Frelinger AL, Du XP, Plow EF, Ginsberg MH. Monoclonal anti-

bodies to ligand-occupied conformers of integrin alpha IIb beta 3

(glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function.

J Biol Chem 1991; 266: 17106–11.

5 Derrick JM, Taylor DB, Loudon RG, Gartner TK. The peptide

LSARLAF causes platelet secretion and aggregation by directly acti-

vating the integrin alpha IIb beta 3. Biochem J 1997; 325: 309–13.

6 Li R, Xie J, Kantor C, Koistinen V, Altieri DC, Nortamo P, Gahm-

berg CG. A peptide derived from the intercellular adhesion molecule-2

regulates the avidity of the leukocyte integrins CD11b/CD18 and

CD11c/CD18. J Cell Biol 1995; 129: 1143–53.

7 Nortamo P, Patarroyo M, Kantor C, Suopanki J, Gahmberg CG.

Immunological mapping of the human leucocyte adhesion glycopro-

tein gp90 (CD18) by monoclonal antibodies. Scand J Immunol 1998;

28: 537–46.

8 Huizinga EG, Van der Plas MR, Kroon J, Sixma JJ, Gros P. Struc-

ture of the A3 domain of human von Willebrand factor: implications

for collagen binding. Structure 1997; 5: 1147–56.

9 Koivunen E,Wang B, Ruoslahti E. Isolation of a highly specific ligand

for the alpha 5 beta 1 integrin from a phage display library. J Cell Biol

1994; 124: 373–80.

10 Koivunen E, Ranta TM, Annila A, Taube S, Uppala A, Joikinen M,

Van Willigen G, Ihanus E, Gahmberg CG. Inhibition of b2 integrin-

mediated leukocyte cell adhesion by leucine-leucine-glycine motif-

containing peptides. J Cell Biol 2001; 153: 905–15.

11 Endenburg SC, Hantgan RR, Lindeboom-Blokzijl L, Lankhof H,

Jerome WG, Lewis JC, Sixma JJ, de Groot PG. On the role of von

Willebrand factor in promoting platelet adhesion to fibrin in flowing

blood. Blood 1995; 86: 4158–65.

12 Pasco S, Monboisse JC, Kieffer N. The alpha 3(IV)185–206 peptide

from noncollagenous domain 1 of type IV collagen interacts with a

novel binding site on the beta 3 subunit of integrin alpha Vbeta 3 and

stimulates focal adhesion kinase and phosphatidylinositol 3-kinase

phosphorylation. J Biol Chem 2000; 275: 32999–3007.

13 Bellavite P, Andrioli G, Guzzo P, Arigliano P, Chirumbolo S,

Manzato F, Santonastaso C. A colorimetric method for the meas-

urement of platelet adhesion in microtiter plates. Anal Biochem 1994;

216: 444–50.

14 Sixma JJ,De Groot PG, Van ZantenH, IJsseldijkM.Anew perfusion

chamber to detect platelet adhesion using a small volume of blood.

Thromb Res 1998; 92: S43–6.

15 Koivunen E, Gay DA, Ruoslahti E. Selection of peptides binding to

the a5b1 integrin from phage display library. J Biol Chem 1993; 268:

20205–10.

16 Burrows L, Clark K, Mould AP, Humphries MJ. Fine mapping of

inhibitory anti-a5 monoclonal antibody epitopes that differentially

affect integrin-ligand binding. Biochem J 1999; 344: 527–33.

17 Ruggeri ZM. Platelets in atherothrombosis.NatMed 2002; 8: 1227–34.

18 Xi X, Bodnar RJ, Li Z, Lam SC, Du X. Critical roles for the COOH-

terminal NITY and RGT sequences of the integrin beta3 cytoplasmic

domain in inside-out and outside-in signaling. J Cell Biol 2003; 162:

329–39.

19 Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS,

Lowell CA, Shattil SJ. Coordinate interactions of Csk, Src, and Syk

kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cyto-

skeleton. J Cell Biol 2002; 157: 265–75.

20 Romijn RA, Bouma B, Wuyster W, Gros P, Kroon J, Sixma JJ,

Huizinga EG. Identification of the collagen-binding site of the von

Willebrand factor A3-domain. J Biol Chem 2001; 276: 9985–91.

21 Vanhoorelbeke K, Depraetere H, Romijn RA, Huizinga EG,

De Maeyer M, Deckmyn H. A consensus tetrapeptide selected by

phage display adopts the conformation of a dominant discontinuous

epitope of a monoclonal anti-VWF antibody that inhibits the von

Willebrand factor-collagen interaction. J Biol Chem 2003; 278: 37815–

21.

22 Wu D, Vanhoorelbeke K, Cauwenberghs N, Meiring M, Depraetere

H, Kotze HF, Deckmyn H. Blood 2002; 99: 3623–8.

23 Hellings M, Engelborghs Y, Deckmyn H, Vanhoorelbeke K, Schi-

phorst ME, Akkerman JWN, de Maeyer M. Experimental indication

for the existence of multiple Trp rotamers in vonWillebrand Factor A3

domain. Proteins 2004; 57: 596–601.

Integrin aIIbb3 activation by VPW peptide 1283

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Chapter VI

General discussion

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Integrins are widely expressed heterodimeric receptors that transfer signals from the

extracellular side to the inside of the cell and vice-versa. Not having any enzymatic properties

of their own, they must rely on conformational changes, aided by the intracellular machinery

to exert their function. The processes of integrin activation as well as subsequent ligand

binding are strictly regulated in time as in the topography of the integrin.

Platelet integrin αIIbβ3 on platelets in suspension has to be activated in order to bind to its

ligands, such as fibronectin, von Willebrand Factor (vWF) and foremost fibrinogen (see table

I-1). Platelet activators invoke different levels of activation of αIIbβ3. It has been shown that

the level of platelet activation is reflected in the quantity of ligand-bound αIIbβ3. The most

potent platelet activators are thrombin and collagen. Platelet activation induces several

signalin proteins that lead to integrin activation; inside-out signalin. Subsequently, activated

integrins are able to bind their ligands. Upon ligand binding a second wave of signalin events

is generated by the integrin: outside-in signalin. The main purpose of outside-in signalin is to

strengthen and stabilize ligand binding. Outside-in signalin has been shown to coincide with

tyrosine phosphorylation of both the integrin as well as well as a plethora of proteins that are

involved in integrin function 1-6

.

When whole blood is perfused over a fibrinogen coated surface αIIbβ3-mediated platelet

adhesion to fibrinogen is observed. The platelet coverage of the surface is independent of the

relative velocity or shear. However at higher shear rates (> 300 s-1

) on top of coverage,

platelet aggregation is observed possibly due to high-shear induced platelet activation.

Platelets bind to and spread on immobilized fibrinogen readily. Spreading is accompanied by

extensive cytoskeletal reorganization in the platelets. Upon fibrinogen immobilization, cryptic

and fibrin specific binding sites become exposed and platelet binding to fibrinogen becomes

independent of the conformational state of αIIbβ3 7. However, the interaction between αIIbβ3

and immobilized fibrinogen is dependent on further signal transduction. When platelets are

treated with agents that induce an elevation of cAMP, platelets become non-responsive to

stimulants and they are in resting state 8,9

. Elevation of cAMP (e.g. with iloprost) in platelets

resulted in abrogation of αIIbβ3-mediated adhesion to immobilized fibrinogen (unpublished

data).

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VI-1 Regulation of the adhesive behaviour of αIIbβ3 is regulated by discrete

regions of the β3 cytoplasmic tail and specific subsequent signalling via

these regions

VI-1.1 E749

ATSTFTN756

of the ββββ3 subunit and its relevance for ligand binding

Intracellular domains of β3 play an important role in optimal αIIbβ3 function. Introduction of a

peptide homologous to the membrane distal part of the β3 subunit has been shown to inhibit

αIIbβ3 mediated adhesion to fibrinogen10

. The membrane distal part of the β3 subunit contains

binding sites for several kinases involved in integrin function. In our study we investigated

the two functional regions present in the membrane distal part by synthesising two peptides,

with a two amino acid overlap that are homologous to the singular peptides mentioned above,

peptide EATSTFTN (E-N) and TNITYRGT (T-T). These peptides were introduced in

streptolysin O permeabilized platelets, after which fibrinogen and fibronectin binding, as well

as signal transduction events could be assessed. To address the question whether observed

effects on ligand binding were due to inside-out or outside in signalling, cells stably

expressing αIIbβ3 and adhering via this integrin to fibrinogen or fibronectin, were

microinjected with peptides (chapter III). For studies with platelets under flow, platelets were

electroporated in the presence of peptide and immediately as whole blood perfused over

fibrinogen (chapter IV). For studying the involvement of the cytoskeleton, platelets were pre-

treated with cytochalasin D. The results indicate that region E-N is important for the

stabilization of ligand binding to αIIbβ3.

Previously, it was shown that introduction of a peptide homologous to E749

ATSTFTN756

,

peptide E-N, and T755

NITYRGT762

, peptide T-T, abrogated fibronectin binding after fifteen

minutes at room temperature8. Further studies by our group on the role of the β3 regions

showed that region E-N stabilizes ligand binding (chapter III). In the first three minutes,

ligand binding in the presence of peptide E-N was normal and then decreased. In contrast,

peptide T-T did not affect ligand binding under three minutes. The decrease in ligand binding

was accompanied by a decrease in FAK tyrosine phosphorylation. When peptide E-N was

microinjected in cells stably expressing αIIbβ3, adhesion to fibrinogen was abrogated. Our data

showed that peptide E-N directly interferes with ligand binding by impairing the stabilization

of ligand binding. This led to the breakdown of existing ligand-integrin complexes. The

activation of the integrin is not hindered by the presence of the peptide, since for the first

three minutes ligand binding was similar to the control situation.

In contrast to platelets in suspension, peptide E-N increased platelet binding to fibrinogen

(measured as coverage) under flow (chapter IV). On closer examination, the increase by the

presence of E-N was mainly due to an increase in adhesion, not spreading.

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Our results with peptide E-N in platelets in suspension and static adhesion to fibrinogen by

αIIbβ3 expressing cells are in line with previous findings 10

. However, the effect of peptide E-N

in platelets perfused over fibrinogen is opposite of the results in platelets in suspension.

The integrin is kept in a low-affinity state by the cytoskeleton, and upon activation actin

turnover is increased which releases the integrin from these constraints. After ligand binding

the integrin engages in cytoskeletal interaction and strong ligand binding is achieved. Low to

medium doses of cytoskeletal inhibitors activate integrins and induce ligand binding, due to

the release of the integrin from the cytoskeleton11

.

Our results showed that cytochalasin D treatment markedly increased platelet adhesion, but

spreading is impaired. This suggests that the actin turnover is sufficient to achieve ligand

binding, which depends on releasing and reattaching to the cytoskeleton.

This is in agreement with previous studies11

. The increase in αIIbβ3-binding to immobilized

fibrinogen was surprising. Since this does not require prior activation of αIIbβ3, binding to

immobilized fibrinogen was expected to be maximal. Thus, our data suggests that the release

from and the reattachment to the cytoskeleton is important in both adhesion to immobilized

fibrinogen, and in fibrinogen binding in suspension 11

.

Finally, since both introduction of E-N into platelets as well as treatment with cytochalasin D

induced an increase in adhesion, it is surprising that E-N reduced the increase caused by

cytochalasin D treatment, when these two agents were combined. This suggests that E-N

sequence is involved in signalling towards the cytoskeleton, supported by the fact that the

spreading is normalized in the case of the combination of E-N and cytochalasin D.

VI-1.2 E749

ATSTFTN756

of the β3 subunit and its relevance for signal transduction

The cytoplasmic tail of the β3 subunit has been shown to be a target of signal transduction as

well as generator of signal transduction. The peptides described above were introduced in

permeabilized platelets in suspension that were subsequently activated. As a measure of

activation tyrosine phosphorylation of proteins of interest was determined. We showed in

chapter III that region E-N signals to FAK.

Not only does region E749

ATSTFTN756

(E-N) contain potential binding sites for kinases but

also potential phosphorylation sites on S (Ser) and T (Thr). It was shown that Thr753 12

is

phosphorylated upon stimulation with thrombin, possibly by PKC 13,14

. The TxT motif is

highly conserved among integrins, and its deletion results in abrogation of ligand binding 15

.

In β2−integrins this motif is also Thr-phosphorylated 16

.

The increase of platelet adhesion by peptide E-N is probably due to taking over the role of the

endogenous region E-N. For this, peptide E-N has to be a substrate for phosphorylation.

Peptides mimicking the β3-tail can be phosphorylated by tyrosine kinases Src, Fyn and Lyn 6.

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Our unpublished data showed that a protein the size of Src-kinase is associated with a Thr753

-

phosphorylated E-N peptide, suggesting that this kinase maybe relevant for αIIbβ3 function.

Tyrosine phosphorylated FAK acts as a major docking site for proteins involved in focal

zones 17

or focal adhesions. The abrogation of FAK tyrosine phosphorylation in the presence

of peptide E-N then contributes to further destabilization of ligand binding.

Our own results in combination with reported data would delineate a model of post ligand

phosphorylation events of the β3-tail, which would start, with the phosphorylation of Thr753

by PKC. This then could be followed by the docking of a Src-kinase on phosphorylated

Thr753

. A model of the putative sequential events is given (figure VI-1A).

PKC-β co-immunoprecipitates with αIIbβ3 upon interaction with either soluble or immobilized

fibrinogen18

, supporting the above proposed order in outside in signalling (figure VI-1A).

However, although PKC-β may be responsible for the phosphorylation of Thr753

in humans,

in a murine PKC knock-out model no effect of single PKC-abrogation was found on αIIbβ3-

mediated ligand binding (I.Hers, personal communication).

Surprisingly, a convincing role for SYK in ligand stabilization was not found. SYK is

phosphorylated upon clustering of integrins and independent of ligand binding 19,20

. Together,

with our data, this suggests that SYK is involved in simultaneous yet ligand independent

signalling or inside-in signalling events rather than post ligand binding. This is further

supported by previous studies 21

in which spatial associations of αIIbβ3, SYK and FAK were

determined by fluorescent imaging. SYK and FAK both interacted with αIIbβ3 but not with

each other. Since there is no interaction between SYK and FAK, it seems unlikely that SYK

would play a role in maintaining ligand binding to αIIbβ3. This is supported by the fact that

SYK knock-out mice do not have a bleeding tendency 20

. However, since for platelets in

suspension a change in affinity rather than a change in avidity is required and SYK is a

substrate to Src-kinase, this suggests SYK as non-pivotal in controlling soluble ligand

binding. In contrast, SYK may be more important for adhesion to immobilized ligands.

As stated, under flow αIIbβ3-mediated adhesion to fibrinogen is regulated differently compared

to the binding of soluble ligand (chapter IV). SYK is implicated in signalling via Vav and

Vav2 towards the small GTPases like rac, rho and CDC42 22

. Each of these GTPases is

involved in different spreading events like formation of lamellipodia, filopodia and formation

of stress fibres, respectively. The fact that introduction of E-N increased rather than decreased

adhesion, suggests that a negative feedback loop is constrained. Peptide E-N provides an

alternative substrate for this feedback loop, liberating the integrin. This signalling may well

include SYK. The increased signalling through SYK may explain the increase in spreading

upon introduction of E-N in cytochalasin D treated platelets by an increase in the activity of

small GTPases. Rho would be a promising candidate for further investigation since the

formation of stress fibres was clearly enhanced (data not shown). The combination of peptide

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Figure VI-1 A: a putative model of the order of signalling events leading to the stabilization of ligand binding and the formation of focal zones.

The integrin is activated, leading to ligand binding. Upon ligand binding Thr753

is phosphorylated which facilitates bindig of Src. These events

lead to the Tyr-phosphorylation of FAK, which signals toward focal adhesions. B: the binding of Src may lead to the Tyr-phosphorylation of

Tyr759

. This in turn may lead to incorporation of the integrin into focal zones, thereby rendering the ligand binding irreversible. The Tyr-

phosphorylation leads to the detachement of β3-endonexin. This is followed by binding of Shc. Please note that figure is not drawn at scale (Fg

is fibrinogen).

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E-N and cytochalasin D decreased adhesion, which supports involvement of SYK with region

E-N and downstream signalling of this region via SYK to the cytoskeleton. Cytochalasin D is

present in sub optimal concentrations thus; an increase in cytoskeletal turnover may overcome

the cytoskeletal impairment. An increase in signalling via SYK and small GTPases could

provide an increase in actin turnover hereby partly restoring cytoskeletal reorganization and

spreading.

Recently there is increasing evidence that talin plays a pivotal role in both integrin activation

as well as linking the integrin to the cytoskeleton 23,24-27

. In previous studies 8, a role for the

talin head binding region in ligand binding in suspension could not be established, and further

studies using our perfusion model did not provide an apparent role for this region (data not

shown). However, our data showed an involvement of the SYK binding site in integrin-

mediated adhesion. Since SYK has been implicated to be involved in talin binding to the

integrin 28

and the integrin binding site of SYK is adjacent to the talin binding site, we cannot

exclude a mutually dependent stabilization of both talin and SYK, which would attenuate

ligand binding to αIIbβ3.

SYK-signalling, and its possible downstream targets rac, rho and cdc42, could be placed close

to region E-N. The down regulation of SYK and hence the abrogation of further signalling

towards rho, rac and cdc42 can be the molecular basis for the decrease in spreading. This

could be in favour of adhesion, as we have shown in chapter IV. We suggest a role for SYK

in signalling towards the small GTPases Rho, Rac and CDC42 in the case of immobilized

ligands. However, this regulation seems to be modulated by a negative feedback loop, since

introduction of E-N leads to an increase in adhesion. SYK would thus keep a balance between

adhesion and spreading via region E-N. Further investigation into the role of SYK and it

relation to region E-N is needed to clarify their respective roles in regulating adhesion and

spreading.

VI-1.3 T755

NITYRGT762

of the β3 subunit and its relevance for ligand binding

The region T755

NITYRGT762

has been shown in the past to be critical for integrin function.

Signalling towards the cytoskeleton is considered to be the main function of this domain. As

discussed above, a larger peptide overlapping this region abolished αIIbβ3-mediated adhesion

to fibrinogen. Since other functional domains were incorporated in this peptide, we chose to

study individual domains as found in the larger peptide. Studies as described above were

performed to assess the function of this domain in platelets in suspension (ligand binding), the

effect on adhesion to fibrinogen of cells expressing αIIbβ3 and in platelets binding to

immobilized fibrinogen under flow. Peptide T-T did not affect ligand binding following

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immediately after platelets stimulation, in contrast to peptide E-N. However, adhesion under

flow to fibrinogen was decreased, which is in agreement with previous studies 8,29

.

Cells transiently expressing the mutated form of αIIbβ3, mutations in the β3 cytoplasmic tail,

deleting Ile757 onwards, showed total abolishment of cell spreading on fibrinogen and

formation of adhesion plaques 30

. The sequence homology in β1, β2 and β3 integrins suggests

that this region is important for integrin activation and cytoskeletal interactions8,31,32

. Deletion

of the cytoplasmic tail starting with Tyr759

showed only partial inhibition of adhesion to

fibrinogen and spreading, indicating that Asp-Ile-Tyr in this motif plays a key role. Even

though deletion Tyr759

did not completely inhibit cell spreading and the formation of adhesion

plaques, both these processes were impaired 30

.

Peptide T-T decreases ligand binding to αIIbβ3 after 15 minutes 8. This was in concordance

with a previous study 10

in which a peptide containing this region interferes with αIIbβ3

mediated adhesion to fibrinogen. In contrast to peptide E-N, that affected ligand binding

shortly after platelet activation (chapter III), peptide T-T interfered only after 15 minutes 8.

Surprisingly, microinjected peptide T-T diffused focal adhesions in cells spreading via αIIbβ3

on fibrinogen rather than abrogating adhesion. This and the results of our studies on platelet

adhesion to fibrinogen under flow, suggest that the formation in focal zones may depend on

region T-T. This is in agreement with previous studies in which formation of focal adhesion

was linked to this region 30

. Even though peptide T-T decreased fibrinogen adhesion,

surprisingly both in cytochalasin D treated and mock treated platelets spreading was

increased.

This suggests that the presence of peptide T-T in cytochalasin D treated platelets restores

signalling towards the cytoskeleton. That would be possible if due to the cytochalasin

treatment region T-T became available for interaction. Peptide T-T would provide in that case

an alternative for endogenous T-T, hereby liberating endogenous T-T. When this is combined

with the fact that peptide T-T affects ligand binding later in time than E-N, it could be that

region T-T modulates focal zones and spreading rather than interfere with ligand stabilization.

Our data suggest that both peptide T-T and cytochalasin D interfere with region T-T of β3-

cytoplasmic tail, but with different outcomes regarding adhesion and spreading.

VI-1.4 T755

NITYRGT762

of the ββββ3 subunit and its relevance for signal

transduction

Peptides E-N and T-T mimicking the membrane distal parts were introduced to study their

effect on αIIbβ3-mediated ligand binding. In a previous study time points earlier than fifteen

minutes were not investigated, and it was shown that peptide T-T inhibited ligand binding at

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this specific time point. Ligand binding to αIIbβ3 is maximal in peptide-untreated thrombin

stimulated platelets after this time point 8. In chapter III we showed that the presence of

peptide T-T had no effect on ligand binding directly after platelet stimulation. These

observations suggest that region T-T becomes involved in later stages of integrin mediated

signalling, and not immediately after ligand binding. All signalling events involving region T-

T may therefore be preceded by signalling events involving region E-N. The rather

immediate involvement of region E-N in ligand binding and maintenance of the ligand bound,

combined with the engagement of region T-T in the formation of focal zones and subsequent

ligand stabilization gives rize to model in figure VI-1B. The sequential events complement

figure VI-1A.

The Tyr in the T-T region of β3 contains an Integrin Cytoplasmic tYrosine (ICY) motif, which

can be found also membrane proximal of that region in N744

PLY. Phosphorylation of the Tyr

in region T-T is necessary for interaction of the β3-tail with Shc and Grb233

. Interaction of

Grb2 with the β3-tail is dependent on dual Tyr-phosphorylation; the second site is provided by

the Tyr of N744

PLY. Mutational analysis 34

showed that Tyr747

and Tyr759

both play a role in

αIIbβ3 mediated spreading and the formation of adhesion plaques, as well as in the binding of

Shc to the β3-cytoplasmic tail35

. The Tyr-phosphorylation site NITY in region T-T overlaps

with the β3-endonexin binding site. Thus, Tyr-phosphorylation of region T-T could abrogate

β3-endonexin binding. This is supported by the fact that β3-endonexin is absent from focal

adhesions, which suggests transient interaction with the β3-tail. The transient interaction

allows further interactions of the β3-tail with the cytoskeleton (chapter IV). Such transient

interactions are often regulated by phosphorylation and dephosphorylation.

Combination of our data supports the hypothesis that region T-T of the β3-tail is involved in

the formation of focal zones or focal adhesions via β3-endonexin rather than be involved

directly in ligand binding itself.

We can conclude from our data that the role of region T-T in the interaction between soluble

fibrinogen and platelets in suspension is less prominent than in the αIIbβ3-mediated adhesion

to immobilized fibrinogen (chapter III). Based on the results obtained by introduction of

peptides E-N and T-T, Tyr-phosphorylation and focal zone formation do not occur at early

stages of integrin activation, and depend on the signalling via region E-N of the β3-

cytoplasmic tail and Thr-phosphorylation, as primary signalling events.

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VI-2 Extracellular regulation of αIIbβ3 function

VI-2.1 von Willebrand Factor and its synergetic role with fibrinogen in αIIbβ3

mediated adhesion

The classic role of vWF is to slow down platelets, which is then followed by arrest on

collagen. Whilst screening an RGD-occupied αIIbβ3 with a phage library for novel αIIbβ3-

protein interactions, a novel integrin recognising a non-RGD tripeptide sequence was found.

The tripeptide sequence VPW was homologous to the VPW present in the vWF A3-domain.

Platelets adhered to an immobilized cyclic VPW containing peptide in a αIIbβ3 -dependent

manner; adhesion of platelets and αIIbβ3-expressing CHO cells to fibrinogen was increased in

the presence of VPW-peptide. In addition, this adhesion to immobilized fibrinogen was faster

in the presence of VPW-peptide. In platelets adhering to fibrinogen, VPW accelerated the

activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. This is

similarly seen in αIIbβ3-expressing CHO cells, where formation of stress fibres was

accelerated by VPW-peptide. In blood from a vWF-deficient individual, VPW increased

platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired

adhesion to vWF deficient in A-3 (Chapter V).

Similarly, a non-RGD-heptapeptide LSARLAF, designed to inhibit fibrinogen binding to

αIIbβ3, has been shown to induce platelet function via αIIbβ3 36

. LSARLAF was designed to

bind next to a presumptive fibrinogen-binding site on the αIIb subunit. Rather than inhibiting

ligand binding to αIIbβ3, binding to this site enhances αIIbβ3 mediated ligand binding followed

by platelet activation.

Not only parts of ligand37,38

or ligand-mimetics 36

but also antibodies binding to an integrin

can induce activation of the integrin and subsequent ligand binding. A class of antibodies

directed against RGD-sequence occupied integrin have been reported in several studies 39,40

,

this in an effort to map conformational changes in the integrin induced by ligand binding.

These antibodies are usually directed against Ligand Induced Binding Site (LIBS). VPW-

phage binds to RGD-sequence occupied αIIbβ3 and shows in that respect similarity to LIBS-

antibodies.

It has been shown that RGD binding inhibits fibrinogen binding to αIIbβ3, yet RGD-peptide

recognizes fibrinogen bound αIIbβ341

. It could be that the RGD-bound state resembles the

ligand bound state the most, which is supported by some studies42

. One such LIBS antibody

(LIBS-6), was able to induce fibrinogen binding both in the presence and absence of RGD-

peptide 43

followed by ligand binding induced signal transduction.

The common molecular mechanism in these ligand binding inducing peptides or antibodies is

that they adhere to binding pockets or in their immediate proximity. For neither cyclic VPW

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peptide nor the VPW present in vWF, the exact binding site on αIIbβ3 is known. VPW is

located in the A3 domain, which is distal to the RGD sequence in the C-terminal part.

In the crystal structure of vWF, the VPW sequence is buried between a β-sheet and an α-helix

of the A3 domain, which is distal to the C-terminal RGD sequence44

.

A monoclonal antibody (82D6A4) directed against the A3 domain of vWF, and specifically

against the sequence SPWR has been reported recently 45

. This sequence could be aligned to

Pro981

Trp982

in the A3 domain, suggesting that VPW is not buried. A study using fluorescence

anisotrophy analysis of Trp 982

shows that two different rotamers of VPW in vWF are

possible: one that agrees with the orientation as found in the crystal structure and another as

an exposed rotamer46

.

VPW-phage was shown to bind to αIIbβ3 in the presence of RGD- peptide. It has been shown 41

that when the RGD-binding site is occupied on αIIbβ3, the fibrinogen binding site is

inaccessible. Recent crystal structures of RGD-occupied αIIbβ3 show similarity to fibrinogen-

mimetic occupied αIIbβ342

. RGD binding to αIIbβ3 in the lower part of the β-propeller sterically

blocks the binding of fibrinogen (-mimetics), an interaction that is in the upper part of the

propeller. VPW may recognize an occupied fibrinogen-binding site and stabilize fibrinogen

binding. Stabilization of fibrinogen binding would explain the increase in platelet adhesion to

immobilized fibrinogen. This stabilization is limited to immobilized fibrinogen since VPW

has no apparent effect on platelets in suspension. VPW could stabilize the binding residues of

the fibrin and immobilized sequence γ316-322

, which is supported by the finding that γ400-411

peptide only partly inhibits platelet binding to a VPW coated surface. An interesting study

would be to use the fibrin specific γ316-322

sequence to study platelet binding to VPW.

Direct αIIbβ3-mediated platelet adhesion to VPW is abrogated by RGD-peptide; in contrast,

binding of VPW-phage to αIIbβ3 is not inhibited by RGD peptide. Purified αIIbβ3 is thought to

be activated upon immobilization 47

. On platelets in suspension, αIIbβ3 is in a resting state, and

hence it could be the conformational state of the integrin influences whether or not RGD-

peptide sterically blocks VPW-αIIbβ3 interaction.

Even though αIIbβ3 binds to VPW-peptide directly, no signal transduction or other platelet

activation is detected. This is only the case in the presence of immobilized fibrinogen. VPW

induces an increase in platelet numbers adhering to fibrinogen and the increase seen in

tyrosine phosphorylation of SYK is higher then would be expected based on the increase in

platelet adhesion. Ligand binding and subsequent cytoskeletal rearrangement leads to

clustering of integrins. Cytoskeletal reorganization enhances tyrosine phosphorylation of

SYK48

. VPW-peptide could facilitate preclustering of the integrin, without generating signal

transduction. Upon ligand binding VPW facilitates the formation of integrin clusters, which

leads to an increase in adhesion as well as SYK tyrosine phosphorylation.

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VPW may shift the integrin from a resting to a preactivated state, possibly by binding to the

vicinity of the fibrinogen-binding site. This hereby facilitates both fibrinogen binding and

integrin clustering, originating from the formation of micro clusters of αIIbβ3.

Our data showed that in the perfusates of vWF-deficient patients, VPW was able to restore

αIIbβ3-mediated adhesion to fibrinogen, suggesting that the RGD in vWF is not necessary for

fibrinogen binding. In contrast, adhesion to collagen was not restored by VPW. On the other

hand, VPW peptide may bind proximal to the RGD-sequence binding site. RGD-peptide

recognizes a conformational state of the integrin in which the ligand-binding interface

resembles a ligand bound or primed state, different from fully activated integrin. This would

mean that VPW binding site is only unmasked in RGD-occupied or fully active integrin (i.e.

purified immobilized αIIbβ3) and VPW binding is deeper in the β-propeller, like the binding

site for RGD-sequence.

The binding of another peptide, CRRETAWAC, was inhibited by RGD even though it binds

to a non-RGD site 49

. Several RGD containing LIBS antibodies induce ligand binding 39,43

.

Further study is needed to map the binding site of VPW on αIIbβ3 and the role of RGD in

recognition of this site. Based on our data, a satisfactory explanation for the RGD-peptide

interference in the binding of platelets to immobilized VPW for this cannot be given.

The role of VPW in vivo remains to be investigated. Even though it is clear that VPW peptide

restored fibrinogen binding in the perfusates of vWF-deficient patients, under normal

circumstances this would mean a constant action by VWP in native vWF. It may well be that

vWF has to be bound to collagen in order to expose fully active VPW. This suggests that

VPW would strengthen the adhesion of platelets to the primary, collagen adhering platelet

layer, hereby providing a tighter basis of a growing platelet aggregate. Pilot studies with

platelets under high, arterial, shear rates showed an increase in aggregation in the presence of

VPW peptide (data not shown).

The VPW sequence may not only provide a novel tool in the determination of the

conformational changes involved in αIIbβ3 activation, but may also be of importance in the

first steps of haemostasis and thrombosis.

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References

1. Brown E, Hogg N. Where the outside meets the inside: Integrins as activators and

targets of signal transduction cascades. Immunol Lett 1996; 54: 189-193.

2. Aota SI, Yamada KM. Integrin functions and signal transduction. Adv Exp Med Biol

1997; 400: 669-682.

3. Aplin AE, Howe A, Alahari SK, Juliani RL. Signal transduction and signal modulation

by cell adhesion receptors: The role of integrins, cadherins, immunoglobulin-cell

adhesion molecules, and selectins. Pharmacol Rev 1998; 50: 197-263.

4. Clark EA, Brugge JS. Integrins and signal transduction pathways: The road taken.

Science 1995; 268: 233-239.

5. Hato T, Pampori N, Shattil SJ. Complementary roles for receptor clustering and

conformational change in the adhesive and signaling functions of integrin αIIbβ3. J Cell

Biol 1998; 141: 1685-1695.

6. Law DA, Nannizzi-Alaimo L, Phillips DR. Outside-in integrin signal transduction -

αIIbβ3-(GP IIb- IIIa) tyrosine phosphorylation induced by platelet aggregation. J Biol

Chem 1996; 271: 10811-10815.

7. Remijn JA, Ijsseldijk MJ, van Hemel BM, Galanakis DK, Hogan KA, Lounes KC, Lord

ST, Sixma JJ, De Groot PG. Reduced platelet adhesion in flowing blood to fibrinogen

by alterations in segment gamma316-322, part of the fibrin-specific region. Br J

Haematol 2002; 117: 650-657.

8. Hers I, Donath J, Litjens PE, Van Willigen G, Akkerman JW. Inhibition of platelet

integrin αIIbβ3 by peptides that interfere with protein kinases and the β3 tail. Arterioscler

Thromb Vasc Biol 2000; 20: 1651-1660.

9. Van Willigen G, Akkerman J-WN. Protein kinase C and cyclic AMP regulate reversible

exposure of binding sites for fibrinogen on the glycoprotein IIB- IIIA complex of human

platelets. Biochem J 1991; 273: 115-120.

10. Liu XY, Timmons S, Lin YZ, Hawiger J. Identification of a functionally important

sequence in the cytoplasmic tail of integrin β3 by using cell-permeable peptide analogs.

Proc Natl Acad Sci USA 1996; 93: 11819-11824.

11. Bennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B. The platelet

cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen. J Biol

Chem 1999; 274: 25301-25307.

12. Lerea KM, Cordero KP, Sakariassen KS, Kirk RI, Fried VA. Phosphorylation sites in

the integrin β3 cytoplasmic domain in intact platelets. J Biol Chem 1999; 274: 1914-

1919.

13. Van Willigen G, Hers I, Gorter G, Akkerman JWN. Exposure of ligand-binding sites on

platelet integrin αIIB/β3 by phosphorylation of the β3 subunit. Biochem J 1996; 314: 769-

779.

-91-

Page 92: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

14. Parise LV, Criss AB, Nannizzi L, Wardell MR. Glycoprotein IIIa is phosphorylated in

intact human platelets. Blood 1990; 75: 2363-2368.

15. Peter K, O'Toole TE. Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1,

CD11a/CD18) cytoplasmic domain/cytoskeleton interaction. J Exp Med 1995; 181: 315-

326.

16. Valmu L, Hilden TJ, Van Willigen G, Gahmberg CG. Characterization of β2 (CD18)

integrin phosphorylation in phorbol ester-activated T lymphocytes. Biochem J 1999;

339: 119-125.

17. Smith A, Carrasco YR, Stanley P, Kieffer N, Batista FD, Hogg N. A talin-dependent

LFA-1 focal zone is formed by rapidly migrating T lymphocytes. J Cell Biol 2005; 170:

141-151.

18. Buensuceso CS, Obergfell A, Soriani A, Eto K, Kiosses WB, Arias-Salgado EG,

Kawakami T, Shattil SJ. Regulation of outside-in signaling in platelets by integrin-

associated protein kinase C beta. J Biol Chem 2005; 280: 644-653.

19. Clark EA, Shattil SJ, Ginsberg MH, Bolen J, Brugge JS. Regulation of the protein

tyrosine kinase pp72syk

by platelet agonists and the integrin αIIbβ3. J Biol Chem 1994;

269: 28859-28864.

20. Law DA, Nannizzi-Alaimo L, Ministri K, Hughes PE, Forsyth J, Turner M, Shattil SJ,

Ginsberg MH, Tybulewicz VL, Phillips DR. Genetic and pharmacological analyses of

syk function in alphaIIbbeta3 signaling in platelets. Blood 1999; 93: 2645-2652.

21. de Virgilio M, Kiosses WB, Shattil SJ. Proximal, selective, and dynamic interactions

between integrin alphaIIbbeta3 and protein tyrosine kinases in living cells. J Cell Biol

2004; 165: 305-311.

22. Miranti CK, Leng L, Maschberger P, Brugge JS, Shattil SJ. Identification of a novel

integrin signaling pathway involving the kinase Syk and the guanine nucleotide

exchange factor Vav1. Curr Biol 1998; 8: 1289-1299.

23. Calderwood DA, Zent R, Grant R, Rees DJG, Hynes RO, Ginsberg MH. The talin head

domain binds to integrin β subunit cytoplasmic tails and regulates integrin activation. J

Biol Chem 1999; 274: 28071-28074.

24. Nayal A, Webb DJ, Horwitz AF. Talin: an emerging focal point of adhesion dynamics.

Curr Opin Cell Biol 2004; 16: 94-98.

25. Tremuth L, Kreis S, Melchior C, Hoebeke J, Ronde P, Plancon S, Takeda K, Kieffer N.

A fluorescence cell biology approach to map the second integrin-binding site of talin to

a 130-amino acid sequence within the rod domain. J Biol Chem 2004; 279: 22258-

22266.

26. Smith JW, Ruggeri ZM, Kunicki TJ, Cheresh DA. Interaction of integrins αVβ3 and

glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for

distinct binding sites. J Biol Chem 1990; 265: 12267-12271.

-92-

Page 93: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

27. Ratnikov BI, Partridge AW, Ginsberg MH. Integrin activation by talin. J Thromb

Haemost 2005; 3: 1783-1790.

28. Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg MH,

Calderwood DA. Talin binding to integrin beta tails: a final common step in integrin

activation. Science 2003; 302: 103-106.

29. Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J

Cell Sci 2000; 113 ( Pt 20): 3563-3571.

30. Ylanne J, Huuskonen J, O'Toole TE, Ginsberg MH, Virtanen I, Gahmberg CG.

Mutation of the cytoplasmic domain of the integrin beta 3 subunit. Differential effects

on cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis. J

Biol Chem 1995; 270: 9550-9557.

31. Hibbs ML, Jakes S, Stacker SA, Wallace RW, Springer TA. The cytoplasmic domain of

the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for

binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated

phosphorylation site. J Exp Med 1991; 174: 1227-1238.

32. Reszka AA, Hayashi Y, Horwitz AF. Identification of amino acid sequences in the

integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. J Cell Biol

1992; 117: 1321-1330.

33. Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin

cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and

platelet function. Nature 1999; 401: 808-811.

34. Ylänne J, Huuskonen J, O'Toole TE, Ginsberg MH, Virtanen I, Gahmberg CG.

Mutation of the cytoplasmic domain of the integrin β3 subunit. Differential effects on

cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis. J Biol

Chem 1995; 270: 9550-9557.

35. Cowan KJ, Law DA, Phillips DR. Identification of shc as the primary protein binding to

the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin

platelet signaling. J Biol Chem 2000; 275: 36423-36429.

36. Derrick JM, Taylor DB, Loudon RG, Gartner TK. The peptide LSARLAF causes

platelet secretion and aggregation by directly activating the integrin αIIbβ3. Biochem J

1997; 325: 309-313.

37. Kotovuori A, Pessa-Morikawa T, Kotovuori P, Nortamo P, Gahmberg CG. ICAM-2 and

a peptide from its binding domain are efficient activators of leukocyte adhesion and

integrin affinity. J Immunol 1999; 162: 6613-6620.

38. Li R, Nortamo P, Kantor C, Kovanen P, Timonen T, Gahmberg CG. A leukocyte

integrin binding peptide from intercellular adhesion molecule-2 stimulates T cell

adhesion and natural killer cell activity. J Biol Chem 1993; 268: 21474-21477.

39. Frelinger AL, III, Du XP, Plow EF, Ginsberg MH. Monoclonal antibodies to ligand-

occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor

affinity, specificity, and function. J Biol Chem 1991; 266: 17106-17111.

-93-

Page 94: Integrin αIIbβ regulation in platelets · lack of exercise, obesity and smoking are commonly acknowledged as risk factors. Most of the risk factors negatively affect the vascular

40. Frelinger AL, III, Cohen I, Plow EF, Smith MA, Roberts J, Lam SC, Ginsberg MH.

Selective inhibition of integrin function by antibodies specific for ligand-occupied

receptor conformers. J Biol Chem 1990; 265: 6346-6352.

41. Hu DD, White CA, Panzer-Knodle S, Page JD, Nicholson N, Smith JW. A new model

of dual interacting ligand binding sites on integrin alphaIIbbeta3. J Biol Chem 1999;

274: 4633-4639.

42. Xiao T, Takagi J, Coller BS, Wang JH, Springer TA. Structural basis for allostery in

integrins and binding to fibrinogen-mimetic therapeutics. Nature 2004; 432: 59-67.

43. Huang MM, Lipfert L, Cunningham M, Brugge JS, Ginsberg MH, Shattil SJ. Adhesive

ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel

protein substrates before phosphorylation of pp125FAK. J Cell Biol 1993; 122: 473-

483.

44. Huizinga EG, Martijn vdP, Kroon J, Sixma JJ, Gros P. Crystal structure of the A3

domain of human von Willebrand factor: implications for collagen binding. Structure

1997; 5: 1147-1156.

45. Vanhoorelbeke K, Depraetere H, Romijn RA, Huizinga EG, De Maeyer M, Deckmyn

H. A consensus tetrapeptide selected by phage display adopts the conformation of a

dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the

von Willebrand factor-collagen interaction. J Biol Chem 2003; 278: 37815-37821.

46. Hellings M, Engelborghs Y, Deckmyn H, Vanhoorelbeke K, Schiphorst ME, Akkerman

JW, De Maeyer M. Experimental indication for the existence of multiple Trp rotamers

in von Willebrand Factor A3 domain. Proteins 2004; 57: 596-601.

47. Yoshimura K, Miyazaki T, Furuyama N, Terashita Z, Fujisawa Y. Binding properties of

fibrinogen receptor GPIIb-IIIa purified from human erythroleukemia cells. Biochem Mol

Med 1995; 56: 166-171.

48. Clark EA, Shattil SJ, Ginsberg MH, Bolen J, Brugge JS. Regulation of the protein

tyrosine kinase pp72syk

by platelet agonists and the integrin αIIbβ3. J Biol Chem 1994;

269: 28859-28864.

49. Mould AP, Burrows L, Humphries MJ. Identification of amino acid residues that form

part of the ligand-binding pocket of integrin alpha5 beta1. J Biol Chem 1998; 273:

25664-25672.

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Summary

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In summary, the studies described in this thesis show that discrete parts of the platelet integrin

αIIbβ3 are involved in discrete ligand binding sub processes. A tripeptide sequence in the A3

domain of vWF is involved in preactivation of αIIbβ3, leading to an increase of both adhesion

to immobilized fibrinogen as well as outside-in signalling. Dependent on whether the

fibrinogen is in suspension or immobilized, two membrane distal parts of the β3-cytoplasmic

tail are involved in:

1) maintaining the ligand bound state with regard to platelets and ligand in suspension

(E-N)

2) attenuating adhesion to immobilized fibrinogen under flow (E-N)

3) maintaining a binding to the cytoskeleton upon ligand binding (T-T)

4) controlling of platelet spreading under flow (T-T).

The basis for the different and opposite roles of the investigated regions may be due to the

fact that under flow the conformational changes of immobilized fibrinogen enable platelet

adhesion without prior activation, whereas, in suspension platelets have to be activated before

ligand binding occurs.

In the future, further analysis of the signal transduction routes affected by the presence of the

peptide in platelets under flow, may give more insight in the difference in the involvement of

the β3-cytoplasmic tail regions compared to platelets in suspension. Of special interest are rac,

cdc42 and foremost rho, being involved in the formation of stress fibres and their upstream

effector SYK. It would be interesting to elucidate the molecular basis of the interaction of

region T-T and the cytoskeleton. For this, studies in which the behaviour upon adhesion to

fibrinogen of individual platelets could give insights in the spreading process. It would also be

interesting to study the activational state of αIIbβ3, which is located on top of the adhered

platelets, in the presence of peptides E-N and T-T. The integrins may be affected by the

presence of the peptides similar to integrins on platelets in suspension. Thr of Tyr-

phosphorylated peptides could aid in the investigation of signaling and ligand binding

processes dependent on phosphorylated residues.

The extracellular parts of αIIbβ3 can be influenced by parts of ligands, for instance VPW in

vWF. In vitro, the VPW sequence may generate a preactivation or micro clustering of αIIbβ3.

VPW increases αIIbβ3-mediated adhesion to immobilized fibrinogen, and increases the

tyrosine phosphorylation of the clustering dependent kinase SYK. The cooperativity of the

binding of RGD, fibrinogen and VPW is necessary for optimal αIIbβ3 adhesion to fibrinogen

under flow.

In vivo vWF could contribute to platelet adhesion on the site of injury by increasing

fibrinogen-αIIbβ3 interaction. One of the questions that remain to be answered is the location

of the VPW-binding site on αIIbβ3. Mapping studies using the fibrin specific γ316-322

sequence

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or antibodies directed against this part of fibrinogen could provide more insight. It would be

interesting to further study the involvement of VPW in micro clustering, by determining if

VPW is able to induce dimers or small multimers of purified αIIbβ3. Perfusion studies at high

shear rate may lead to better understanding of the effect of VPW on platelets layering

(aggregation). Whether or not VPW preactivates αIIbβ3, it will be difficult to investigate,

however, electron microscopy of VPW-αIIbβ3 complexes may show the prevalence of this

complex for an intermediate state αIIbβ3 closer resembling the fully activated conformation.

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Samenvatting voor niet-ingewijden

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Het menselijk bloed, evenals het bloed van vele gewervelde dieren, bevat een mechanisme

dat er voor zorgt dat uitstromen van bloed na verwonding beperkt wordt. Men noemt dit

mechanisme haemostase.

Rondom bloedvaten bevindt zich een laag collageen. Als een bloedvat door verwonding

beschadigd wordt, heeft het bloed vrijelijk contact met deze laag collageen. De kleinste van

alle bloedcellen, het bloedplaatje of trombocyt, heeft op het celoppervlak receptoren die aan

collageen kunnen binden. Echter, de snelheid van de trombocyt is te hoog om direct vanuit de

bloedstroom aan collageen te binden. Een eiwit wat in overvloed aanwezig is in de

bloedstroom, von Willbrand factor, biedt uitkomst. Von Willebrand factor bindt aan collageen

en aan trombocyten, en door zijn structuur fungeert het als klittenband; trombocyten remmen

al rollende over von Willebrand factor af en binden door middel van receptoren stabiel aan

collageen.

Er is een aantal collageenreceptoren op trombocyten beschreven. Een belangrijke

overeenkomst is dat op ze allemaal door de celmembraan heensteken. Hiermee kunnen

signalen van de buitenkant (extracellulair) naar de binnenkant (intracellulair) doorgegeven

worden en vice-versa . Als er aan de buitenkant binding met collageen optreedt, leidt dat tot

grote veranderingen in de trombocyt. Men noemt dit activatie.

Activatie is een verzameling van processen, en heeft tot doel er voor te zorgen dat

trombocyten gaan samenklonteren (aggregeren) en zo de wond afdichten. De trombocyt

specifieke receptor, de fibrinogeen receptor of integrine αIIbβ3 speelt bij aggregatie de

belangrijkste rol. Het integrine αIIbβ3 heeft een extracellular en een (klein) intracellulair deel.

Onder normale omstandigheden kan fibrinogeen niet aan αIIbβ3 binden. De receptor is in rust

of niet-geactiveerd. Als een trombocyt geactiveerd raakt, wordt ook αIIbβ3 geactiveerd

Hierdoor verandert de structuur van αIIbβ3 en is binding van fibrinogeen uit het bloed aan de

trombocyt mogelijk. Zo kan er een heel netwerk van trombocyten aangelegd worden. Dit

wordt een trombus genoemd en deze dekt de wond af.

Tegelijkertijd met dichten van de wond een ander mechanisme in het bloed geactiveerd. Dit

systeem zet fibrinogeen om in fibrine. Als fibrinogeen omgezet wordt in onoplosbaar fibrine

versterkt dit de trombus. Trombine is het enzym dat fibrinogeen omzet in fibrine. Tevens is

trombine een sterke activator van bloedplaatjes. Trombine zet trombocyten aan tot wat heet

‘clot retractie’, het persen van de trombus waardoor deze zicht verdicht, waarmee de

wonddichting maximaal wordt.

Belangrijk voor de functie van de trombocyt is het cytoskelet. Men kan dit vergelijken met het

menselijk skelet. Het cytoskelet is een systeem van eiwitten, dat een netwerk vormen zodat

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de trombocyt zijn vorm krijgt zoals we die kennen: een discusvorm. Na activatie vindt er

intensieve reorganisatie van het cytoskelet plaats zodat een trombocyt over een zo groot

mogelijke afstand uitgesmeerd wordt en zich daarna ineentrekt voor de clotretractie.

In rust is een groot deel van het integrine αIIbβ3 verbonden via het intracellulaire deel aan het

cytoskelet. Bij activatie wordt αIIbβ3 losgekoppeld van het cytoskelet en kan het aan

fibrinogeen binden. Na fibrinogeenbinding vindt een stevige verankering plaats aan het

cytoskelet.

Het onderzoek dat in dit proefschrift is beschreven, heeft zich gericht op de mechanismen die

de trombocyt gebruikt om integrine αIIbβ3 te openen (activatie) en de daaropvolgende

fibrinogeenbinding. Door trombocyten lek te maken kunnen stukjes van αIIbβ3 (peptiden) in

gebracht worden. Hiermee is het mogelijk om de functies van de overeenkomende stukken in

het aanwezige integrine αIIbβ3 nader te bekijken. Het blijkt dat gedeelten van het

intracellulaire deel van αIIbβ3 op verschillende wijzen betrokken zijn bij het openen en het

open houden van de fibrinogeen-bindingsplaatsen op αIIbβ3. Afhankelijk van de

omstandigheden waarin de trombocyt zich bevindt, in supsensie of in een nagebootste

bloedstroom, zijn deze domeinen in staat om bij te dragen aan stabilisatie van de

fibrinogeenbinding of een verankering aan het cytoskelet. De verankering aan het cytoskelet

leidt ook tot een stabilisatie van de fibrinogeenbinding. Maar uit de resultaten blijkt dat er in

de nagebootste bloedstroom ook een beperking van de ligandbinding plaatsvindt: de binding

kan vergroot worden in de aanwezigheid van een peptide.

Het eiwit ‘Focal Adhesion Kinase’ (FAK) speelt hierin een belangrijke rol. De activatie van

FAK kan herleid worden tot een bepaald gedeelte van het intracellulaire deel van αIIbβ3 voor

trombocyten in suspensie. Voor trombocyten in de nagebootste bloedstroom heeft dit FAK

activerende domein wellicht ook een andere rol. Waarschijnlijk is het zo dat activatie van

eiwitten betrokken bij de cytoskeletaire reorganisatie ook terug te voeren is op dit domein,

maar verder onderzoek in de nagebootste bloedstroom zou dit moeten verduidelijken.

Vanuit het intracellulaire deel gezien, is integrine functie in suspensie en in de nagebootste

bloedstroom sterk verschillend van elkaar. Overeenkomende processen vinden deels in een

andere volgorde plaats en deels is de betrokkenheid van een proces anders. Zo speelt hetzelfde

intracellulaire deel van de fibrinogeen receptor onder verschillende omstandigheden een

andere rol. Hierdoor worden ook vervolgprocessen anders aangestuurd of beïnvloed. Hierdoor

kan een trombocyt afhankelijk van de omstandigheden anders functioneren.

Zoals boven beschreven, bestaat de grootste rol van von Willebrand factor uit het afremmen

van trombocyten zodat stabiele hechting in het wondgebied mogelijk is. In dit proefschrift

wordt een nieuwe rol voor von Willebrand factor beschreven: het vergroten van de adhesie

aan met fibrinogeen bedekte oppervlakken.

Drie naast elkaar liggende aminozuren (VPW) in het deel van von Willebrand factor dat de

interactie met collageen verzorgd blijken een directe interactie aan te gaan met de αIIbβ3.

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Hierdoor wordt er iets in αIIbβ3 verandert waardoor binding aan fibrinogeen onder bepaalde

condities (het moet zich in een oppervlak bevinden) vergemakkelijkt wordt. Dit zien we terug

in het feit dat meer plaatjes aan een fibrinogeen oppervlak hechten in de aanwezigheid van

een peptide wat bestaat uit VPW.

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Nawoord

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Uiteindelijk is het af! Na een paar jaar onderbreking door een carrière buiten de wetenschap

ben ik na het uitkomen van het laatste artikel (hoofdstuk VI in dit proefschrift), afgelopen

zomer begonnen met het aaneenschrijven van de verschillende hoofdstukken.

Onconventioneel en onorthodox zult u zeggen –enkelen zeiden het reeds- maar ik denk dat

wetenschap per definitie gebaat is bij non-conventionaliteit en onorthodoxie, mits dit tot een

goed resultaat leidt. Uiteindelijk geldt de door mij in de laatste tijd veel gehoorde uitspraak:

‘the proof of the pudding is in eating it’.

Toen ik, net afgestudeerde biochemicus/tumor-immunoloog met filosofische achtergrond,

geruime tijd geleden begon als OIO aan dit project dacht ik nog dat er maar 1 waarheid zou

kunnen zijn aangaande de werking van een bepaald mechanisme. Zoals u kunt lezen in de

diverse hoofdstukken heb ik proefondervindelijk vast moeten stellen dat dit niet altijd waar is,

al kan men veel groeperen onder de parapluie-theorie. Maar de details, dus wanneer het echt

interessant wordt, laten zich niet zo makkelijk vangen. Hiermee heb ik een beetje afstand

moeten nemen van de door mij geliefde Cartesiaanse methode, en heeft het postmoderne

relativisme ook zijn intrede gedaan bij mij en ook wel in dit proefschrift: een

wetenschappelijk model is niet waar of onwaar, maar het is een adequate verklaring of

voorspelling van de verschijnselen. Men kan een analogie trekken tussen het

wetenschappelijk model en een kaart. Kaarten zijn er in soorten (ook al gaan ze over het

zelfde gebied), en ze helpen ons uitstekend om daar te komen waar we willen, zonder de

noodzaak te geloven dat de weergave exact gelijk is aan de werkelijkheid. Dit pluralisme lijkt

in eerste instantie de wetenschappelijke discussie dood te slaan, maar kan mijns inziens juist

ieder bestaand idee juist een extra dimensie geven. Voor mijzelf heeft het in ieder geval een

hele andere, niet onprettige, kijk op mijn eigen onderzoek gegeven.

Het bovenstaande als achtergrond. Op mijn tijd in het AZU kijk ik met veel plezier terug. Dan

komen we aan bij het bedanken van hen die een bijdrage geleverd hebben bij de

totstandkoming van dit proefschrift:

Mijn promotor Jan-Willem Akkerman: beste chef, de afgelopen jaren heb ik veel van je

geleerd. Terugkijkend moet ik opmerken dat we wel erg vaak zeer genoeglijke aan congressen

gerelateerde etentjes in ‘la douce France’ gehad hebben. Hoe je dat doet moet je me nog maar

eens leren!

Mijn begeleider Gijsbert van Willigen: beste Gijsbert: met zijn tweeën op een viermansproject

was niet altijd even makkelijk: werk te over, en het was vaak moeilijk kiezen. Ik kijk met

groot plezier terug op de verschillende periodes dat we samen in Helsinki aan het werk waren.

Daar zijn zeer mooie resultaten en ideeën uit voort gekomen, naast on-Hollandse-winter-

gezelligheid (een bijzondere mix van weinig daglicht, mobiele telefonie, sauna, drank en 1-

kamerhuisvesting voor ons 2).

Gertie Gorter: beste Gertie: je vele tips en gedegen kennis van het experimenteel verleden

waren een ware hulp. Als ‘dagelijkse contacten’ via de MDD en onze bezigheden op het

radionuclidenlab hebben ons toch vaak meer dan alleen een glimlach op het gezicht getoverd.

Mijn studenten: Irma Lutters: beste Irma, je zus werkte reeds bij ons, maar in ongeveer niets

bleek je op haar te lijken. Hoe rustig zij, hoe zeer aanwezig jij was. Jij was een soort co-

project van Gijsbert en mij, maar ik heb een hele leuke en productieve tijd met je gehad. Je

hebt de pilot experimenten van de perfusie gedaan, en dat heeft toch een mooi artikel

opgeleverd. Daarnaast deelden we een soort absurde humor, en dat was, zeker toen we

geisoleerd op het nova-zemblalab zaten, vaak erg verfrissend. De tweede lading: de miekes:

the original Mieke: beste Mieke, jij kwam na Irma voor 6 maanden stage lopen en dat beviel

ons beiden zo goed dat we er nog 6 achteraan geplakt hebben. Na 6 maanden perfusie ben je

verder gegaan met ‘vis’-proeven. Helaas is het niet gelukt dat af te ronden, hoewel die

experimenten toch een mooi inzicht in de affiniteit van Src voor αIIbβ3 gegeven hebben. Naast

de proeven was het ook nog buiten werktijd erg gezellig, al duurde de werktijd soms wel tot

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na 10en. Ook daar was je niet te benauwd voor, mits ik je dan wel naar huis chauffeerde.

Mieke-2: Yvonne van Willigen: beste Yvonne, naast de rustige Mieke was jij extreem goed

gebekt, maar goed ook want je verschijning deed menig mannenoog jouw richting uit gaan,

wat vaak tot gevat commentaar van jouw kant leidde. Je was met Mieke samen een zeer

spontaan en apart team, en jullie hebben mij zeer veel verder geholpen! Ton: ook bij jou

kwamen direct beelden van excellente lunches en zondagochtenden met XO-cognac in me

naar voren, maar daarnaast hebben we (ja ook met drank erbij) toch hele goede discussies

gehad over toekomstige experimenten. Ik wil wel die kamer tussen jou en Cora in de

Memorial Wing. Gelukkig werkten we ook nog wel eens…..

De dames van het secretariaat (later secretariaten): Joukje, Carin, Elvira en Irma: het non-

bostonian ‘Cheers’ op de afdeling. Niet alleen voor vele organisatorische zaken maar ook een

niet-wetenschappelijk informatieuitwisseling en vooral veel simpele humor. Carin: zelfs nu

kom ik nog graag langs voor je schaterende lach!

Mijn vrienden hebben allen op hun eigen manier mij in de loop der jaren gesteund.

Verscheidene van mijn collegae mag ik tot mijn vrienden rekenen. Enkele van mijn vrienden

zou ik graag hier willen noemen:

José: here is looking at you kid, we will always have Lyon. Jouw uitermate kritische en open

kijk op het onderzoek heeft mij veel geleerd en gebracht (met 1/3 V bindingsproeven doen

heeft me heel wat minder peptide gekost). Daarnaast wordt en werd jouw gewillige oor zeer

op prijs gesteld. Bovendien was het soms gewoon heerlijk hilarisch (pellets op aparte plekken,

woordspelingen etc….) Ik ben blij dat we goede vrienden zijn geworden.

Chris: hoe kleiner de man hoe groter het ego, en je komt er nog mee weg ook. Jouw warme en

persoonlijke aandacht heeft me altijd erg geraakt. Jouw gespot ook. Het waren heerlijk tijden

toen we nog samen IP’s deden en op vrijdag naar de Beurs gingen voor een zinloze avond.

Een zekere duopresentatie op een feestje zal ik nooit vergeten.

Relou: wat hebben we niet meegemaakt? En welke cocktails hebben we nog niet gedronken?

Bekvechtend in het lab gevolgd door gierend-van-het-lachen rennen in Tivoli. Bedankt voor

alle goede en hilarische gesprekken en je serieuze aandacht voor mijn onderzoek. En toch

vind ik dat je die muizenproeven had moeten doen. Snel naar de Japanner.

Edwin: je bent een vrolijke noot en goede vriend geworden in de tijd die ik je nu ken, en je

hebt in het lab maar ook over landsgrenzen bijgedragen aan de feestvreugd. Ik heb overigens

nog geen wijn gezien met het nieuwe firmalabel. Waarvan akte.

Els/Ellen: kordaat en eigenzinnig. Ook zonder bier op mag je wel een keer op mijn schoot

zitten hoor. Jammer dat de EDSO-vleugel nooit van de grond is gekomen.

Pia: the non-silent Finn. I cherish our friendship that is based literally on adhering platelets.

Thank you for the many discussions we had on our subjects and the razor sharp criticism.

Special thanks for the wise words on ‘how to write a thesis’ and your comments.

Tina & Krien: voor jullie typische Groningse stijl van steun: zwijgend maar o zo aanwezig.

Beer en Eef: Beer: onze gesprekken hebben me gesteund het toch door te zetten. Het

gereedkomen is in niet geringe mate aan jouw steun te wijten. Eef: bedankt voor de

literatuurservice, de correcties en commentaren en voor de peptalk als het wat duister werd. Ik

ben enorm blij dat je mijn paranimf wilt zijn.

Koessie & Pepke: jullie hebben me altijd vrij gelaten in mijn keuzes en me daarin gesteund.

Koessie:jouw soms grote persoonlijke offers om mij te doen slagen hebben veel voor mij

betekend. Ik ben er trots op dat je mijn paranimf bent.

Ron: je bent in mijn leven gekomen in het laatste hectische halfjaar van mijn onderzoek. Jouw

kalmte en rust hebben een positief effect op mij gehad. Jouw liefde was een grote bron van

steun en troost als het minder ging. Het is af, nu kunnen eindelijk die dozen met mijn

literatuur archief de logeerkamer uit.

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Curriculum Vitae

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De schrijver van dit proefschrift werd op 5 mei 1971 geboren te Maasbracht. Na het behalen

van het Gymnasium-β-diploma (1989) aan het Bisschoppelijk College te Weert werd

begonnen met de studie Scheikunde aan de toenmalige Katholieke Universiteit Nijmegen (nu

Radboud Universiteit Nijmegen). Het doctoraaldiploma behaalde hij in 1997, met als

uitgebreide hoofdrichting Biochemie (prof.dr. W.J. van Venrooij en dr. R.M. Hoet) en als

uitgebreide nevenrichting Tumorimmunologie/haematologie (prof.dr. Y. van Kooijk en dr.

M.E. Binnerts). Van 1997-2002 was hij werkzaam als hij werkzaam voor de Nederlandse

Organisatie voor Wetenschappelijk Onderzoek (NWO) als onderzoeker in opleiding bij de

vakgroep Haematologie van het Universitair Medisch Centrum Utrecht. Gedurende deze

periode werd onder begeleiding van prof. dr. J.W.N. Akkerman en dr. G. van Willigen het

onderzoek verricht dat tot dit proefschrift heeft geleid. De schrijver werkte van 2003-2005 als

product support officer bij Roche Diagnostics Nederland. Heden werkt de schrijver als

marketing communications specialist, global marketing lysosomal storage diseases, Genzyme

Therapeutics, en is gestationeerd in Naarden.

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