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Integrin αIIbβ3 regulation in platelets
Pieter E.M.H. Litjens
Cover: Platelets, spreading and adhering.
Yvonne van Willigen
ISBN 90-902-1072-5
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
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.
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
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
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-
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-
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-
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-
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-
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-
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-
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-
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.
-15-
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-
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.
-17-
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-
-18-
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
.
-19-
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.
-20-
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
.
-21-
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.
-22-
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).
-23-
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-
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-
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-
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-
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-
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-
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-
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-
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.
-32-
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-
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
-34-
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.
-35-
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-
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-
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.
-38-
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:
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
-39-
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.
-41-
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.
PLATELETS 313
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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 ).
-43-
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
PLATELETS 315
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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-
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-
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-
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.
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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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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-
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-
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
� 2005 International Society on Thrombosis and Haemostasis
-69-
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
� 2005 International Society on Thrombosis and Haemostasis
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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
� 2005 International Society on Thrombosis and Haemostasis
-71-
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
� 2005 International Society on Thrombosis and Haemostasis
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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
� 2005 International Society on Thrombosis and Haemostasis
-73-
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
� 2005 International Society on Thrombosis and Haemostasis
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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
� 2005 International Society on Thrombosis and Haemostasis
-75-
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
� 2005 International Society on Thrombosis and Haemostasis
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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
� 2005 International Society on Thrombosis and Haemostasis
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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
-79-
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).
-84-
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
-88-
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.
-89-
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-
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-
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-
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
-102-
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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