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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2645-2657
REVIEW ARTICLE
Integrin Signaling: The Platelet Paradigm
By
Sanford J. Shattil,
Hirokazu Kashiwagi, and
Nisar Pampori
From the Departments of Vascular Biology and Molecular and
Experimental Medicine, The Scripps Research Institute, La Jolla, CA.
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INTRODUCTION |
ADHESION IS REQUIRED for cell
growth, differentiation, survival, and function. Nowhere is this more
evident than in the response to tissue injury, where vascular damage
triggers reparative processes, such as hemostasis, inflammation, and
wound healing. These processes depend on a coordinated series of cell
adhesion and migration events by platelets, leukocytes, and vascular
cells for their successful execution.1 Cell adhesion is
mediated by a structurally diverse group of plasma membrane receptors,
each exhibiting specialized ligand-binding properties that are needed
for specific tasks in the injury response. For example, when blood
flows through a damaged blood vessel, leukocytes slow down and roll on
the endothelial surface as a consequence of the interaction of
appropriate sialyl Lewis X-rich membrane glycoproteins on the
leukocytes with selectins on the endothelial cells.2,3
Platelets also roll under conditions of high shear on perturbed
endothelium4 as well as on denuded vascular surfaces, in
the latter case through interactions of the platelet glycoprotein (GP)
Ib-V-IX complex with von Willebrand factor (vWF) in the subendothelial
matrix.5 Once the rolling process has slowed down these
blood cells, they come to an abrupt stop at the right place through
regulated interactions between integrin adhesion receptors and either
counter-receptors on endothelial cells or adhesive proteins in the
matrix.2,5 Integrins also mediate responses necessary for
eventual completion of the injury response, including leukocyte
transmigration and platelet aggregation.2,6
Although adhesion receptors rightfully deserve this moniker, any
implication that they are simply cellular velcro is incorrect. Most, if
not all, adhesion receptors engage in a dialogue with the extracellular
and intracellular milieus. Integrins are a case in point. Cells often
regulate ligand binding to integrins through a process known as
inside-out signaling or integrin activation. Furthermore, once
integrins have become occupied and clustered by their ligands, they can
transmit information into cells. These outside-in signals collaborate
with signals originating from growth factor receptors and other plasma
membrane receptors to regulate a host of anchorage-dependent cellular
functions. One of the best studied cases of integrin signaling involves
 IIb 3, an integrin of particular
significance to hematologists because it is required for aggregation
and adhesive spreading of platelets during hemostasis (Fig 1). The purpose of this review is to describe the
platelet paradigm of integrin signaling and to emphasize the advances
and gaps in our understanding of this process and place it into
clinical perspective. We have tried to cite authoritative reviews
whenever possible to provide interested readers with additional sources of primary references. Several excellent general reviews of
integrins7-12 and platelet biochemistry13,14
are available.
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| Fig 1.
Integrin signaling in hemostasis. Platelet adhesion to
the damaged vessel wall is initiated by platelet rolling, an
integrin-independent event mediated by binding of GP Ib-V-X
to vWF (left panel). Subsequent stationary adhesion and primary
platelet aggregation require inside-out signaling through and
ligand binding to IIb3 (center panel). Full platelet spreading, aggregation, and effective hemostatic plug
formation also require outside-in signaling through
IIb3 (right panel).
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WHAT IS INTEGRIN SIGNALING? |
IIb3 consists of a two-chain subunit
bound noncovalently to a single-chain subunit. Each subunit spans
the platelet membrane once. The N-terminus and most of the remainder of
each subunit are extracellular, and the membrane-spanning domain is connected to a short C-terminal cytoplasmic tail consisting of 20 amino
acid residues in IIb and 47 residues in
3. Electron microscopy of heterodimers shows an
N-terminal globular head connected to two C-terminal
stalks.15,16 Although the atomic structure of
IIb3 is not known, biochemical, genetic,
and molecular modeling studies indicate that ligand binding is
primarily a function of the globular heads.17 Because
ligand binding is regulated by signals from within the platelet and
also triggers platelet responses, mechanisms must exist to propagate
information back and forth between the cytoplasmic tails and the
globular heads. This overall process is referred to as integrin
signaling.
A didactic distinction is often made between inside-out and outside-in
signaling. Inside-out signaling denotes those reactions initiated by
the binding of one or more agonists to their plasma membrane receptors,
leading to the conversion of IIb3 from a low-affinity/avidity receptor to a high-affinity/avidity receptor. This
conversion has profound consequences in that it determines whether
IIb3 can engage soluble adhesive ligands,
such as fibrinogen and vWF, which contain a classical integrin
recognition sequence, Arg-Gly-Asp. These multivalent ligands can
function as bridges between receptors on adjacent platelets, thus
allowing platelet aggregation to proceed.18 Because
IIb3 can diffuse laterally within the
plasma membrane, inside-out signaling can have two distinct components
that are often difficult to distinguish in practice: (1) affinity
modulation, which implies a structural change intrinsic to the
heterodimer that results in a greater strength of ligand binding; and
(2) avidity modulation, which implies a change in the functional
affinity of the interaction between receptor and ligand due
to chelate or rebinding effects.19 One
plausible way that the latter could occur is through integrin clustering within the plane of the plasma membrane
(Fig 2).
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| Fig 2.
What is integrin signaling? In this cartoon of the
platelet membrane interface, arrows labeled 1a and 1b denote inside-out signaling pathways and arrow 2 denotes outside-in signaling
pathways. Inside-out signaling increases the affinity (1a)
and avidity (1b) of IIb3 for ligands such
as fibrinogen. Affinity modulation is depicted hypothetically here as a
signal-induced rotation of the 3 subunit to generate and
unmask fibrinogen binding sites in the extracellular domains of
IIb3. Outside-in signaling triggers a
number of postligand binding events and these require cooperative signaling between IIb3 and agonist
receptors (hashed arrow).
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Outside-in signaling denotes reactions initiated by integrin ligation
and clustering, and these must be coordinated with signals emanating
from other plasma membrane receptors (eg, growth factor, cytokine, and
G-protein-linked receptors).10,20,21 Integrin signals help
to regulate a host of postligand binding events, the particular pattern
varying with the cell and the integrin. Postligand binding events
regulated by IIb3 in platelets include the stabilization of large platelet aggregates, platelet spreading, granule secretion, clot retraction, and possibly platelet procoagulant activity.22
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INSIDE-OUT SIGNALING IN PLATELETS |
Resting platelets contain about 80,000 surface copies of
IIb3, with additional pools of
IIb3 in the membranes of -storage granules and the open-canalicular system.23 The binding of
soluble ligands to IIb3 can be detected
within seconds of platelet activation, and it reaches a steady-state
within minutes.18,24 Although ligand binding is at first
reversible, it becomes progressively irreversible.22
Purified IIb3 can bind fibrinogen with a
stoichiometry up to a 1:1, but the stoichiometry may be lower in
platelets. Although ligand-binding to surface-expressed
IIb3 is essential for initial, primary
platelet aggregation, the internal pools of
IIb3 can become exposed after cell
activation and participate in the secondary phase in which larger
platelet aggregates are formed. In fact, the -granule membrane pool
of IIb3 may already be complexed with
fibrinogen stored within these granules.25 Should the
surface pool of receptors on resting platelets become unavailable to
bind ligand, as for example after infusion of a function-blocking
antibody,26 the -granule pool may be able to support
platelet aggregation.27
Affinity versus avidity modulation.
Platelets and other cells use a conformational switch mechanism
(affinity modulation) and receptor clustering (avidity modulation) to
regulate ligand binding to integrins, and the relative contribution of
each varies with the integrin and the cell type.28,29
Ligand binding studies alone cannot usually distinguish between these two mechanisms. Available evidence indicates that the initial, reversible phase of ligand binding to
IIb3 is due to affinity modulation,
whereas the irreversible phase may be due to several factors, including
(1) ligand-induced changes intrinsic to the receptor (perhaps analogous
to those responsible for induced fit between an antibody and
antigen)30,31; (2) receptor clustering32-35;
and (3) receptor internalization.36 In addition,
thrombospondin and other substances released from -granules during
secretion may bind to fibrinogen and/or
IIb3 and stabilize the ligand-receptor interaction.37 An initial conformational switch mechanism
is consistent with the rapid and selective binding of a monovalent, ligand-mimetic antibody Fab fragment to
IIb3 after platelet activation.38 Moreover, fluorescence resonance energy
transfer studies using monoclonal antibodies bound to extracellular
domains of IIb and 3 show that platelet
activation is associated with a change in the relative orientation of
the subunits.39 Electron micrographs of purified
IIb3 have shown that fibrinogen binding to the globular head of the integrin can be triggered by interaction of
a monoclonal antibody with the membrane-proximal stalk of
3.40 This proves that a long-range
conformational change can be propagated along the integrin, a possible
requirement for affinity modulation. It is logical to assume that
IIb3 also clusters into multimers in
response to cytoskeletal changes during platelet activation. A
subpopulation of IIb3 already is linked
to the membrane skeleton in resting platelets, and there is a wholesale
redistribution of this integrin to the F-actin core cytoskeleton during
platelet activation.14 However, major cytoskeletal
rearrangements do not seem necessary for initial high-affinity ligand
binding to IIb3, because inhibitors of
actin polymerization have a minimal effect on reversible ligand
binding, although they do have a more substantial effect on
irreversible binding.34
The structural changes in IIb3
responsible for interconversion between low- and high-affinity states
are not known. One model posits that the signaling reactions triggered
by platelet agonists cause some modification of the integrin
cytoplasmic tails which is then propagated to the extracellular domains
to effect ligand binding (Fig 2). Recent progress has been made in
understanding the kinds of structural changes in the globular head of
IIb3 that may be required. Based on model
building and the functional effects of mutations,
Springer41 has proposed that the N-terminal region of
IIb (and other integrin subunits) conforms to the shape of a -propeller with seven blades oriented radially and pseudosymmetrically around a central axis and parallel to the plasma
membrane. The ligand binding interface would lie on the top surface of
the propeller (Fig 3). Tozer et
al42 have proposed that a second ligand binding site is
located in an N-terminal region of 3 that bears homology
with an I-domain, which is, ironically, a ligand-binding module of
approximately 190 amino acids inserted within certain subunits (but
not IIb). Crystallographic analyses of I domains from
L and M show an / fold consisting
of seven -helices packed against a six-stranded -sheet. At one
end of the -sheet is a cation binding MIDAS motif implicated in
ligand binding (Fig 3).43,44
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| Fig 3.
A model depicting the potential changes in the
extracellular domains of IIb3 that are
required for high-affinity ligand binding. The top left panel shows an
overhead view of the proposed -propeller domain within the
N-terminal segment of IIb,41 and the top
right panel shows the crystal structure of an I-domain,43 a
homologue of which appears to be present in the N-terminal segment of
3.42 Open circles denote divalent cations
and asterisks denote regions presumed to be directly involved in ligand
binding. Thick ribbons are strands of -sheet, and coiled ribbons are
-helices (adapted from Chothia and Jones172 with
permission, from the Annual Review of Biochemistry, Volume 66, ©1997, by Annual Reviews Inc). The bottom panels illustrate
potential changes in these domains as
IIb3 is converted from a resting state
(left panel) to an activated state (right panel). (Adapted from Loftus
and Liddington.45 Adapted and reproduced from The Journal
of Clinical Investigation, 1997, Vol. 99, pp. 2302, by copyright
permission of The American Society for Clinical Investigation.)
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Based on this information, Loftus and Liddington45 have
proposed a model for the conformational switch in
IIb3 that provides a good framework for
further studies. It predicts that, in resting platelets, the
I-domain-like region in 3 is incapable of binding ligand, but it occludes the ligand binding site in
IIb.45 Platelet activation would then induce
ligand binding by (1) causing a conformational change in the
3 I domain to expose its ligand binding site and (2)
changing the orientation of the subunits to unmask the ligand binding
site in IIb (Fig 3). This implies that the receptor may engage discontinuous regions of the ligand, consistent with the fact that each fibrinogen monomer is multivalent with respect to
IIb3. For example, the C-terminus of the
fibrinogen  chain is essential for initial binding of the soluble
ligand to platelet IIb3,46,47
but one or both of the Arg-Gly-Asp sites in the A chain may provide
secondary points of attachment needed for tighter binding. These A
sites may also assume importance when the fibrinogen is immobilized on
a surface or converted to fibrin.48,49
Reactions that initiate and propagate inside-out signaling.
Inside-out signaling involves reactions that (1) initiate and propagate
the flow of information from agonist or antagonist receptors to
integrin proximal effectors and (2) directly effect integrin activation
or deactivation. Currently, only a broad outline of these reactions can
be provided.
Inside-out signaling is triggered by many excitatory agonists, some of
which, including thrombin, ADP, epinephrine, and thromboxane A2, bind to heptahelical receptors coupled to
heterotrimeric () G proteins.13,50-52 In the case
of some of these agonists, one consequence important for inside-out
signaling is activation of phospholipase C by the
activated subunit of Gq, resulting in hydrolysis of
phosphatidylinositol and production of the second messengers,
diacylglycerol and IP3. Mouse platelets that have been
rendered null for Gq undergo shape change but fail to
aggregate in response to thrombin, ADP, or a thromboxane A2
receptor agonist, and the mice exhibit prolonged tail bleeding
times.53 Occupancy of many G-protein-coupled platelet
receptors also leads to rapid activation of nonreceptor protein
tyrosine kinases, including Src, Syk, and Pyk2 (also known as RAFTK or
CAK).54,55 Although the mechanism by which G proteins
couple to tyrosine kinase cascades in platelets has not been
characterized, the net result is tyrosine phosphorylation of a number
of proteins, including phospholipase C ,56
Vav (a guanine nucleotide exchange factor for the Rac GTP-ase), and
cortactin (a cortical actin-binding protein).54,57,58 A
role for tyrosine phosphorylation-dephosphorylation in integrin activation is suggested by observations that tyrosine kinase inhibitors partially block fibrinogen binding and platelet aggregation, whereas inhibitors of protein tyrosine phosphatases trigger platelet
activation.14,54,59 Furthermore, mouse platelets that have
been rendered null for Syk show a modest reduction in fibrinogen
binding in response to ADP and epinephrine.60 Additional
support for a tyrosine phosphorylation-integrin activation connection
comes from studies of three agonist receptors that are not known to be
coupled to G proteins.
The Fc receptor, FcRIIA, contains an immune receptor tyrosine
activation motif (ITAM) in its cytoplasmic tail. When the receptor is
clustered by aggregated Igs, two tyrosines in the ITAM are phosphorylated by a Src family kinase, enabling Syk, which contains tandem SH2 domains, and possibly other proteins with SH2 domains to
bind. This leads to Syk activation and, eventually, platelet aggregation.61,62 Surprisingly, a similar scheme may
underlie platelet aggregation by collagen. Collagen supports platelet
adhesion indirectly by helping to retain vWF in the vessel
wall.63,64 It also supports adhesion directly through
interactions with integrin 21 and GP IV
(CD36).65,66 However, none of these interactions is
sufficient to trigger platelet activation and recent evidence implicates a 62-kD membrane protein, GP VI, in this
process.67 GP VI exists in a complex with FcR, a 14-kD
ITAM-containing signaling subunit.68,69 Collagen or
suitable triple helical collagen-like peptides bind to GP VI,
stimulating tyrosine phosphorylation of FcR, activation of Syk, and
tyrosine phosphorylation and activation of phospholipase
C2.70,71 A similar chain of events is
observed if platelets are incubated with convulxin, a snake venom
protein specific for GP VI,72 or if GP VI is cross-linked
by an antibody.67 Collagen-induced platelet aggregation is
absent in patients deficient in GP VI as well as in mice null for Fc
or Syk.67,73 Interestingly, activation of Syk and
IIb3 is also triggered by platelet
adhesion to vWF, despite the fact that the relevant adhesion receptor, GP Ib-V-IX, does not possess ITAMs.74
Thus, one common feature of most agonists that activate
IIb3 is their ability to induce
(poly)phosphoinositide hydrolysis and formation of IP3 and
diacylglycerol, either through Gq and phospholipase
C or through tyrosine kinases and phospholipase C.51 IP3 stimulates an increase in
cytoplasmic free Ca2+, but this alone is not sufficient to
activate IIb3.75 A
Na+/Ca2+ exchanger may change the sensitivity
of IIb3 to agonists, but it is not clear
how.76 Activation of conventional PKC isoforms by
diacylglycerol (or by phorbol myristate acetate) leads to activation of
IIb3, a response blocked by PKC
inhibitors.77 A prominent PKC substrate in platelets is
pleckstrin, a protein with two PH domains,78 but no
functional link between pleckstrin and
IIb3 has been established.
Parenthetically, MARCKS proteins are prominent PKC substrates in some
cells, and they have been implicated in integrin-dependent spreading of
macrophages.79
Another signaling molecule that has been implicated in integrin
function is phosphatidylinositol 3-kinase (PI 3-kinase), which converts
PtdIns(4)P and PtdIns(4,5)P2 to the
3-phosphorylated phosphoinositides, PtdIns(3,4)P2
and PtdIns(3,4,5)P3, respectively.80,81 Two
isoforms of this enzyme have been described in platelets, p85/p110 and
p110.80,82 The catalytic activity and subcellular localization of the p110 subunit of p85/p110 are regulated through protein-protein interactions of p85, which contains a Bcr homology domain, SH3 domain, two SH2 domains, and proline-rich sequences. Accordingly, this isoform would be expected to be regulated by proteins
that become tyrosine phosphorylated in response to platelet agonists.
Consistent with this idea, PI 3-kinase can be coprecipitated with Src
and Syk from lysates of activated platelets.83,84 The
catalytic activity of p110, which exists in a complex with a 101-kD
protein, is regulated by G protein subunits.80,82
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are
membrane-embedded and transduce signals, at least in part, by binding
proteins via their specific PH or SH2 domains and recruiting them to
the membrane. Examples include the PH domain-containing proteins, Akt,
a serine-threonine kinase, and TIAM-1, a Rho family guanine nucleotide
exchange protein; or the SH2 domain-containing proteins,
phospholipase C and Src.85 In platelets, thrombin
stimulates a rapid and transient increase in
PtdIns(3,4,5)P3 and a later increase in
PtdIns(3,4)P2. Whereas inhibitors of PI 3-kinase partially
block agonist-induced activation of IIb3
and platelet aggregation,80 it has been suggested that
3-phosphorylated phosphoinositides function more to stabilize fibrinogen binding than to initiate it.86 Furthermore,
accumulation of PtdIns(3,4)P2 is dependent on fibrinogen
binding to IIb3,80 more
consistent with a role for this particular lipid in outside-in signaling. Indeed, PtdIns(3,4)P2 has been implicated in
mediating actin assembly within filopodia and in stimulating a late
phase of pleckstrin phosphorylation in activated
platelets.80,87,88 One possible link between PI 3-kinase,
PKC, and affinity modulation of IIb3 is
the observation that 3-phosphorylated phosphoinositides can
activate certain atypical and novel isoforms of PKC, some of which are
present in platelets.14,85
Members of the Ras superfamily of GTP-ases have also been implicated in
integrin function. Platelets contain several members of the Ras (H-Ras,
Rap1a) and Rho (cdc42, Rac1, RhoA) families and proteins that regulate
their GDP/GTP contents: guanine nucleotide exchange factors, guanine
nucleotide dissociation inhibitors, and GTP-ase activating
proteins.14 Rac1 regulates thrombin-induced actin
polymerization in platelets.89 It has been suggested that RhoA regulates platelet aggregation based on the observation that C3
exoenzyme, an inhibitor of Rho, blocks aggregation responses to
thrombin.90 However, C3 exoenzyme has no effect on affinity modulation of IIb3 or primary platelet
aggregation, although it does block the formation of focal adhesions
and stress fibers during platelet spreading on
fibrinogen.91 Thus, one function of Rho A may be to
regulate cytoskeletal organization and integrin clustering rather than
integrin affinity.92 Expression of activated R-Ras
increases integrin-mediated adhesion in some cells,93 but
its presence in platelets has not been demonstrated. Thrombin stimulation of platelets causes GTP loading of H-Ras in a PKC-dependent manner and of Rap1 in a Ca2+-dependent
manner.94,95 Platelets contain several potential H-Ras
effectors, including PI 3-kinase and Raf-1, and thrombin induces
activation of MAP (ERK2) kinase in platelets, possibly through the
classical H-Ras pathway.96 When overexpressed in CHO cells,
activated H-Ras or Raf-1 can suppress integrin
activation.97 In platelets, the converse is true: the ERK2
response to thrombin is dampened by fibrinogen binding and
aggregation.98
Pathways that inhibit IIb3 are just as
important as those that activate it. Prostaglandin I2
produced by endothelial cells is a potent platelet activation and
aggregation inhibitor that binds to a specific Gs-coupled heptahelical
receptor, thereby activating adenylyl cyclase and cyclic AMP-dependent
protein kinase (PKA). Platelet aggregation is also inhibited by nitric
oxide, which is synthesized by both endothelial cells and platelets and activates soluble guanylyl cyclase (PKG).99 The importance
of the nitric oxide inhibitory pathway in vivo is shown by two brothers with a defect in the bioavailability of nitric oxide, heightened platelet reactivity to agonists, and a history of cerebrovascular events.100 One common substrate of PKA and PKG is VASP, a
50-kD protein that localizes to focal adhesions and regulates actin dynamics.101 The phosphorylation of VASP on specific serine
residues by agents that activate PKA or PKG correlates with inhibition of platelet aggregation.102 However, PKA and PKG are likely
to exert their inhibitory effects on IIb3
at several levels of stimulus-response coupling, implying that more
than one effector of these serine-threonine kinases is
involved.13,102,103 CD39 is an ecto-ADPase on endothelial
cells that may be an important regulator of platelet responses to
ADP.104 Platelets express PDGF -receptors and store PDGF
in their -granules. Incubation of platelets with PDGF dampens
subsequent aggregation responses to excitatory agonists.105
Reactions that effect inside-out signaling.
The conformational switch necessary for ligand binding to
IIb3 could be regulated by intracellular
molecules that bind to the cytoplasmic tails of the integrin or by
integrin-associated membrane proteins. Evidence directly implicating
the cytoplasmic tails in affinity modulation comes from studies of
naturally occurring and experimental integrin mutations, from analyses
of IIb3 function in heterologous
expression systems, and from identification of integrin tail-binding
proteins. In addition, several membrane proteins have been reported to
form complexes with IIb3 or other integrins.
The sequences of the cytoplasmic tails of IIb and
3 are shown in Fig 4. Two
patients with genetic abnormalities in the 3 cytoplasmic
tail provide living examples of the importance of this tail in integrin
signaling. Both exhibit bleeding disorders of mild to moderate severity
due to variant thrombasthenia: despite near-normal levels of
IIb3, their activated platelets bind
neither fibrinogen nor aggregate. One of these individuals exhibits a point mutation in 3 (3
S752P),106 the other exhibits a deletion of the 39 C-terminal residues from the 3 tail
(3  724).107 In each case, the
profound defect in activation of IIb3 can be recapitulated by expressing the recombinant mutant in CHO
cells.107,108 Transfection studies in CHO cells and in a
B-lymphocyte cell line have shown that other mutations in the
cytoplasmic tails also affect IIb3
affinity.109-113 These results can be summarized as follows. Wild-type IIb3 exists in a
default low-affinity state in these cells, but two different classes of
tail alterations lead to a constitutive high-affinity state. One
involves deletions or mutations of specific membrane-proximal residues
in the IIb or 3 tails, causing the
receptor to remain in a high-affinity state even if cellular ATP is
depleted. The other class involves replacement of the
IIb tail with certain other tails (eg,
5 or 6), but in this case the receptor
reverts to a low-affinity state upon depletion of ATP. This class of
energy-dependent, high-affinity mutants can also be inhibited by
overexpression of isolated 3 cytoplasmic tail chimeras,
suggesting that integrin affinity is being regulated by titratable
intracellular factors.114 This idea is supported by the
observation that IIb3-dependent adhesion of a megakaryocytic cell line is inhibited by cellular incorporation of
peptides derived from the membrane-distal region of the
3 tail.115
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| Fig 4.
Amino acid sequences of the cytoplasmic tails of
IIb and 3. The space inserted into each
sequence arbitrarily separates the N-terminal membrane-proximal and
C-terminal membrane-distal regions, the significance of which is
discussed further in the text. Numbered residues are as in the
full-length integrin subunit.
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These results suggest a working model in which the membrane-proximal
portions of the IIb and 3 tails normally
interact, possibly in part through a salt bridge, to form a hinge
through which signals impacting on membrane distal tail residues are
propagated across the membrane to modulate receptor affinity. Certain
membrane-proximal mutations or deletions break this hinge, leaving the
receptor in a permanent high-affinity state. Membrane-distal tail
residues might regulate receptor affinity in several ways: In
unstimulated cells, the IIb tail might bind a negative
regulator or interact with the 3 tail in such a way as
to prevent the action of a positive regulator. In stimulated cells, a
change in these relationships would either relieve the negative
constraint or trigger the function of the positive regulator. This
model predicts close but dynamic interactions between the
IIb and 3 tails. In fact, synthetic peptides derived from these tails do interact in
vitro.116,117
A number of proteins have been shown to bind directly to integrin
cytoplasmic tails, at least in vitro (Table
1), but there is no evidence yet that the endogenous forms of any of
these proteins modulate integrin affinity in cells. One such protein,
3-endonexin, binds selectively to the 3
tail and is present in platelets.118,119 Overexpression of
a 3-endonexin fusion protein in CHO cells increases the
affinity state of IIb3 and causes
fibrinogen-dependent cell aggregation.120 Although some of
the other proteins listed in Table 1 are present in platelets, it is
not known if they influence IIb3
affinity. Two proteins listed in the table may not be relevant to
IIb3, but they provide potential novel
links between integrins and cellular signaling pathways. Cytohesin-1
binds selectively to the 2 integrin tail and when
overexpressed in T lymphocytes, it increases cell adhesion through
L2.121 Cytohesin-1 contains a
PH domain, which binds 3-phosphorylated phosphoinositides, and a sec 7 domain, which binds the 2 tail and possesses guanine nucleotide exchange activity for a small GTP-ase,
ARF.122,123 One serine-threonine kinase, p59ILK
has been shown to bind to integrin tails, to inhibit
1-mediated cell adhesion, and to promote
anchorage-independent cell cycle progression and growth of epithelial
cells.124 These studies suggest that, in some cases,
integrins may be direct targets of protein kinases, phosphatases, or
GTPases. In this regard, the 3 cytoplasmic tail does
become phosphorylated on serine, threonine, and tyrosine residues in
thrombin-stimulated platelets.125-127 However, the
stoichiometry and functional significance of these events are not
clear. Furthermore, the tyrosine phosphorylation of 3 is
dependent on platelet aggregation; therefore, it is more likely to play
some role in outside-in signaling.
Several transmembrane or GPI-linked membrane proteins have been shown
to either coimmunoprecipitate with integrins or colocalize with them by
fluorescence microscopy (Table 2). These
associations may be direct or indirect, and several are relevant to
IIb3. CD47, also known as
integrin-associated protein, spans the platelet plasma membrane five
times and coimmunoprecipitates with 3
integrins.128 So far, no direct role for CD47 in
IIb3 function has been demonstrated, either in platelets or in the CHO cell model system.129
However, CD47 may function as a costimulatory agonist receptor in
platelets because binding of thrombospondin to CD47 leads to activation of IIb3 in a Gi-dependent
manner.130,131 CD98, a type II transmembrane protein
implicated in neutral amino acid transport and viral syncytia formation, was recently identified in a genetic screen by its ability
to complement dominant suppression of
IIb3 activation in CHO cells, but its
abundance in platelets is not known.132 CD9, a member of
the tetraspanin family of transmembrane proteins, colocalizes with
IIb3 in platelet -granule membranes
and filopodia.133 Antibodies to CD9 can stimulate platelet
aggregation in an Fc receptor-independent manner.134
However, tetraspanins may exist in multimolecular complexes, and the
interaction of CD9 with IIb3 may not be
direct. There is similar uncertainty in interpreting the reported
associations of integrins with caveolin or other proteins, such as Src
family kinases, that may become part of large complexes within
lipid-rich membrane microdomains.135,136 Thus, the
functional and physical relationships between
IIb3 and other proteins remain a fertile
area for further investigation.
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OUTSIDE-IN SIGNALING IN PLATELETS |
Platelet functions regulated by outside-in signaling.
Signaling through IIb3 determines the
extent to which platelets spread on a vascular matrix containing vWF or
fibrinogen and their resistance to detachment from the
matrix.5,137 Similarly, outside-in signals triggered during
platelet aggregation or spreading promote granule secretion and
secondary aggregation. Consequently, outside-in signals are a
determinant of the ultimate size of a hemostatic plug or a pathological
thrombus (Fig 1). The retraction of a fibrin clot also involves
outside-in signals because it represents the interaction of both fibrin
and the actin cytoskeleton with IIb3 and
the contraction of actin-myosin.138-140 Under certain conditions, even the development of platelet procoagulant activity due
to scrambling of membrane phospholipids is dependent, in part, on
events subsequent to platelet aggregation.141
The temporal and spatial hierarchy of outside-in signaling.
Outside-in signaling is initiated at localized regions of cell matrix
and cell-cell contact. In the platelet, it is triggered by
ligand-induced oligomerization of IIb3,
because only multivalent ligands are capable of inducing the
signal.38,142 Signaling is propagated by interactions
between integrin cytoplasmic tails, signaling molecules, and structural
cytoskeletal proteins, including vinculin, talin, and -actinin. The
initial signaling reactions foster continued assembly of the complex by
promoting protein-protein interactions, actin polymerization, and
cytoskeletal reorganization. Complex assembly continues until the
supply of new components is exhausted or a set of inhibitory signaling
reactions takes over, at which time the complex may even disassemble.
The platelet has provided a good model system to study outside-in
signaling and cytoskeletal reorganization in the absence of nuclear
signaling (Fig 5).143-145
Within seconds of binding soluble or immobilized fibrinogen or vWF,
platelets extend filopodia coincident with activation of Syk and
tyrosine phosphorylation of substrates of 50 to 68 kD and 140 kD.87,143,145,146 Shortly thereafter, the platelets begin
to flatten out or form microscopic aggregates. At this intermediate
stage, there is detectable activation of pp60Src, and
clusters of IIb3 are discernible by
immunofluorescence microscopy on the basal surfaces of the adherent
cells. Recent studies in CHO cell transfectants indicate that
fibrinogen binding can trigger activation of Syk in a manner that is
independent of ITAMs and actin polymerization, due to a combination of
autophosphorylation and phosphorylation by Src.147
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| Fig 5.
Outside-in signaling through
IIb3 in platelets, emphasizing the
sequential nature of the process. First, agonists induce affinity
modulation and ligand binding promotes integrin clustering (1). Second,
the ligated and clustered integrins trigger early outside-in signaling
events, such as activation of Syk and Src (2). Although not shown, this
may be associated with filopodial extension. Finally, activation
and/or cytoskeletal translocation of FAK, protein tyrosine phosphatases
(PTP), and many other important enzymes (Etc.) occurs, coincident with
their assembly into mature focal adhesions that are connected to actin
stress fibers (3).
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|
Platelet spreading on fibrinogen or vWF reaches a maximum after several
minutes, during which time the platelets display microscopic vinculin
clusters connected to F-actin cables, the platelet equivalent of focal
adhesions.91,146,148 Full spreading or aggregation is
associated with activation of the tyrosine kinase,
pp125FAK, and tyrosine phosphorylation of additional
substrates, including proteins of 101 and 105 kD, Tec (a tyrosine
kinase that contains a PH domain), and SHIP, an SH2 domain-containing
inositol 5-phosphatase.9,145,149,200 None of these changes
occur if spreading or aggregation is blocked. Eventually there is a
decrease in tyrosine phosphorylation of many substrates, due to
cytoskeletal recruitment and activation of protein tyrosine
phosphatases (eg, PTP-1B and SHP-1), and cleavage of protein tyrosine
kinases by calpain.54,146,150-152
FAK provides a well-studied example of how integrin-associated
signaling complexes may assemble.9,12,153 It contains a central catalytic domain flanked by N- and C-terminal domains. Subcellular localization of FAK is dictated by a focal adhesion targeting region in the C-terminal domain and possibly by a binding site for integrin tails in the N-terminal domain. FAK undergoes autophosphorylation at Y397 in response to cell adhesion,
providing a docking site for the SH2 domain of Src and possibly
PI-3-kinase. Src phosphorylates FAK at additional tyrosine residues,
creating sites for interaction of the adapter, Grb2. FAK also complexes
through proline-rich motifs in the C-terminal domain with the SH3
domains of the adaptors, p130cas and paxillin, and a Rho
GTPase-activating protein, GRAF. Indeed, p130cas and paxillin are
phosphorylated by the FAK/Src complex, enabling the recruitment of even
more proteins. FAK-null mice die in fetal life and, ex vivo, their
fibroblasts form focal adhesions but migrate poorly.154 In
platelets, activation of FAK requires both
IIb3 ligation and agonist receptor
occupancy, the latter being required to provide costimulatory signals
through Ca2+ and PKC.155
Studies of naturally occurring and experimentally induced mutations and
deletions in IIb3 provide strong evidence
for involvement of the IIb and 3
cytoplasmic tails in outside-in signaling.107,108,156 However, many fundamental questions remain. What is the role of tyrosine phosphorylation of the 3 tail in response to
platelet aggregation?127 Can certain protein or lipid
kinases and phosphatases couple directly to
IIb3? What are the effectors of Syk, Src,
and FAK? Whereas tyrosine phosphorylation is an early event in
outside-in signaling, there is an impressive and growing list of other
protein and lipid kinases, phosphatases, phospholipases, and GTP-ases that redistribute to the IIb3-rich core
cytoskeleton or become activated during platelet aggregation and
spreading.89,144,157-161,201 How are the functions of so
many proteins and their effectors integrated into the highly
coordinated response to platelet adhesion?
| |
PERSPECTIVE |
What are the practical implications of integrin signaling? Integrin
cytoplasmic tail mutations in patients with variant thrombasthenia prove that integrin signaling is required for hemostasis, but these
patients are very rare. However, other individuals with unexplained
platelet aggregation defects are encountered more frequently. Some of
these suffer from inherited defects, others from acquired disorders
that affect platelet function. Once an aggregation defect has been
established in the clinical laboratory, further evaluation can be
facilitated by conducting flow cytometry analyses of platelets, even in
whole blood. Fluorophore-conjugated reagents are available to
quantitate platelet surface antigens, including activation-specific
antigens (eg, P-selectin) and epitopes (eg, activated or
ligand-occupied IIb3).162
This allows facile categorization of the abnormality as either an
IIb3 activation defect or a postligand
binding defect.163 In the case of an activation defect, the
subsequent work-up can focus on specific agonist receptors and
biochemical pathways responsible for inside-out
signaling.164-166 In the case of a postligand
binding defect, the work-up can focus on the possibility of storage
pool disease or an abnormality in pathways triggered by integrin
ligation.107,167,168 We speculate that the spectrum of
clinical abnormalities in integrin signaling might even include
inappropriate increases in IIb3 function. For example, several dominant mutations introduced experimentally into
the IIb or 3 cytoplasmic tails result in
constitutive activation of the receptor, as discussed above. If such
mutations were to occur naturally, they might be responsible for some
cases of unexplained, chronic thrombocytopenia or even represent a risk
factor for arterial thrombosis.
Interest in IIb3 has expanded beyond the
realm of the hematologist because of the development of pharmacological
inhibitors of ligand binding to IIb3 for
prophylaxis and therapy of arterial thrombosis.169,170
Abciximab, a chimeric mouse-human antibody that blocks ligand binding
to IIb3, is already licensed for use as
adjunctive therapy in patients undergoing coronary angioplasty, and
additional parenteral and orally active compounds are now |
Introduction
References