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.2,3Platelets 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 αIIbβ3, 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 biochemistry13,14are available.

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 αIIbβ3 (center panel). Full platelet spreading, aggregation, and effective hemostatic plug formation also require outside-in signaling through αIIbβ3 (right panel).

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 αIIbβ3 (center panel). Full platelet spreading, aggregation, and effective hemostatic plug formation also require outside-in signaling through αIIbβ3 (right panel).

WHAT IS INTEGRIN SIGNALING?

αIIbβ3 consists of a two-chain α subunit bound noncovalently to a single-chain β 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 αIIb and 47 residues in β3. Electron microscopy of heterodimers shows an N-terminal globular head connected to two C-terminal stalks.15,16 Although the atomic structure of αIIbβ3 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 αIIbβ3 from a low-affinity/avidity receptor to a high-affinity/avidity receptor. This conversion has profound consequences in that it determines whether αIIbβ3 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 αIIbβ3 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).

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 αIIbβ3 for ligands such as fibrinogen. Affinity modulation is depicted hypothetically here as a signal-induced rotation of the β3 subunit to generate and unmask fibrinogen binding sites in the extracellular domains of αIIbβ3. Outside-in signaling triggers a number of postligand binding events and these require cooperative signaling between αIIbβ3 and agonist receptors (hashed arrow).

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 αIIbβ3 for ligands such as fibrinogen. Affinity modulation is depicted hypothetically here as a signal-induced rotation of the β3 subunit to generate and unmask fibrinogen binding sites in the extracellular domains of αIIbβ3. Outside-in signaling triggers a number of postligand binding events and these require cooperative signaling between αIIbβ3 and agonist receptors (hashed arrow).

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 αIIbβ3 in platelets include the stabilization of large platelet aggregates, platelet spreading, granule secretion, clot retraction, and possibly platelet procoagulant activity.22 

INSIDE-OUT SIGNALING IN PLATELETS

Resting platelets contain about 80,000 surface copies of αIIbβ3, with additional pools of αIIbβ3 in the membranes of α-storage granules and the open-canalicular system.23 The binding of soluble ligands to αIIbβ3 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.22Purified αIIbβ3 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 αIIbβ3 is essential for initial, primary platelet aggregation, the internal pools of αIIbβ3 can become exposed after cell activation and participate in the secondary phase in which larger platelet aggregates are formed. In fact, the α-granule membrane pool of αIIbβ3 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 α-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.28,29Ligand binding studies alone cannot usually distinguish between these two mechanisms. Available evidence indicates that the initial, reversible phase of ligand binding to αIIbβ3 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 α-granules during secretion may bind to fibrinogen and/or αIIbβ3 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 αIIbβ3 after platelet activation.38 Moreover, fluorescence resonance energy transfer studies using monoclonal antibodies bound to extracellular domains of αIIb and β3 show that platelet activation is associated with a change in the relative orientation of the subunits.39 Electron micrographs of purified αIIbβ3 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 β3.40 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 αIIbβ3 also clusters into multimers in response to cytoskeletal changes during platelet activation. A subpopulation of αIIbβ3 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 αIIbβ3, 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 αIIbβ3responsible 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 αIIbβ3 that may be required. Based on model building and the functional effects of mutations, Springer41 has proposed that the N-terminal region of αIIb (and other integrin α subunits) conforms to the shape of a β-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 β3 that bears homology with an I-domain, which is, ironically, a ligand-binding module of approximately 190 amino acids inserted within certain α subunits (but not αIIb). Crystallographic analyses of I domains from αL and αM show an α/β fold consisting of seven α-helices packed against a six-stranded β-sheet. At one end of the β-sheet is a cation binding MIDAS motif implicated in ligand binding (Fig 3).43,44 

Fig. 3.

A model depicting the potential changes in the extracellular domains of αIIbβ3 that are required for high-affinity ligand binding. The top left panel shows an overhead view of the proposed β-propeller domain within the N-terminal segment of αIIb,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 β3.42 Open circles denote divalent cations and asterisks denote regions presumed to be directly involved in ligand binding. Thick ribbons are strands of β-sheet, and coiled ribbons are α-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 αIIbβ3 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.)

Fig. 3.

A model depicting the potential changes in the extracellular domains of αIIbβ3 that are required for high-affinity ligand binding. The top left panel shows an overhead view of the proposed β-propeller domain within the N-terminal segment of αIIb,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 β3.42 Open circles denote divalent cations and asterisks denote regions presumed to be directly involved in ligand binding. Thick ribbons are strands of β-sheet, and coiled ribbons are α-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 αIIbβ3 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.)

Based on this information, Loftus and Liddington45 have proposed a model for the conformational switch in αIIbβ3 that provides a good framework for further studies. It predicts that, in resting platelets, the I-domain–like region in β3 is incapable of binding ligand, but it occludes the ligand binding site in αIIb.45 Platelet activation would then induce ligand binding by (1) causing a conformational change in the β3 I domain to expose its ligand binding site and (2) changing the orientation of the subunits to unmask the ligand binding site in αIIb (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 αIIbβ3. For example, the C-terminus of the fibrinogen γ chain is essential for initial binding of the soluble ligand to platelet αIIbβ3,46,47but one or both of the Arg-Gly-Asp sites in the Aα chain may provide secondary points of attachment needed for tighter binding. These Aα 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 (αβγ) G proteins.13,50-52 In the case of some of these agonists, one consequence important for inside-out signaling is activation of phospholipase Cβ by the activated α 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 A2receptor 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 CAKβ).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 Cγ,56Vav (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, FcγRIIA, 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 α2β1 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 FcRγ, 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 FcRγ, activation of Syk, and tyrosine phosphorylation and activation of phospholipase Cγ2.70,71 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 Fcγ or Syk.67,73 Interestingly, activation of Syk and αIIbβ3 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 αIIbβ3 is their ability to induce (poly)phosphoinositide hydrolysis and formation of IP3 and diacylglycerol, either through Gq and phospholipase Cβ or through tyrosine kinases and phospholipase Cγ.51 IP3 stimulates an increase in cytoplasmic free Ca2+, but this alone is not sufficient to activate αIIbβ3.75 A Na+/Ca2+ exchanger may change the sensitivity of αIIbβ3 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 αIIbβ3, 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 αIIbβ3 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)P2and PtdIns(3,4,5)P3, respectively.80,81 Two isoforms of this enzyme have been described in platelets, p85/p110 and p110γ.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 p110γ, which exists in a complex with a 101-kD protein, is regulated by G protein βγ 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 Cγ 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 αIIbβ3and 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 αIIbβ3,80 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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 are just as important as those that activate it. Prostaglandin I2produced 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 αIIbβ3at 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 α-receptors and store PDGF in their α-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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 or other integrins.

The sequences of the cytoplasmic tails of αIIb and β3 are shown in Fig 4. Two patients with genetic abnormalities in the β3 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 αIIbβ3, their activated platelets bind neither fibrinogen nor aggregate. One of these individuals exhibits a point mutation in β33S752P),106 the other exhibits a deletion of the 39 C-terminal residues from the β3 tail (β3 Δ 724).107 In each case, the profound defect in activation of αIIbβ3 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 αIIbβ3affinity.109-113 These results can be summarized as follows. Wild-type αIIbβ3 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 αIIb or β3 tails, causing the receptor to remain in a high-affinity state even if cellular ATP is depleted. The other class involves replacement of the αIIb tail with certain other α tails (eg, α5 or α6), 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 β3 cytoplasmic tail chimeras, suggesting that integrin affinity is being regulated by titratable intracellular factors.114 This idea is supported by the observation that αIIbβ3-dependent adhesion of a megakaryocytic cell line is inhibited by cellular incorporation of peptides derived from the membrane-distal region of the β3 tail.115 

Fig. 4.

Amino acid sequences of the cytoplasmic tails of αIIb and β3. 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.

Fig. 4.

Amino acid sequences of the cytoplasmic tails of αIIb and β3. 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.

These results suggest a working model in which the membrane-proximal portions of the αIIb and β3 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 αIIb tail might bind a negative regulator or interact with the β3 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 αIIb and β3 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 (Table1), but there is no evidence yet that the endogenous forms of any of these proteins modulate integrin affinity in cells. One such protein, β3-endonexin, binds selectively to the β3tail and is present in platelets.118,119 Overexpression of a β3-endonexin fusion protein in CHO cells increases the affinity state of αIIbβ3 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 αIIbβ3affinity. Two proteins listed in the table may not be relevant to αIIbβ3, but they provide potential novel links between integrins and cellular signaling pathways. Cytohesin-1 binds selectively to the β2 integrin tail and when overexpressed in T lymphocytes, it increases cell adhesion through αLβ2.121 Cytohesin-1 contains a PH domain, which binds 3-phosphorylated phosphoinositides, and a sec 7 domain, which binds the β2 tail and possesses guanine nucleotide exchange activity for a small GTP-ase, ARF.122,123 One serine-threonine kinase, p59ILKhas been shown to bind to integrin β tails, to inhibit β1-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 β3 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 β3 is dependent on platelet aggregation; therefore, it is more likely to play some role in outside-in signaling.

Table 1.

Integrin Tail-Binding Proteins

Protein  Integrin Tail Partner  Notable Features Reference  
Calreticulin  α* Expression correlates with integrin-mediated cell adhesion; present in many subcellular locations.  173-176  
F-actin  α2 only Structural cytoskeletal protein  177  
Calcium- and integrin-binding protein (CIB)  αIIb only  Sequence homology to calcineurin B; contains 2 EF-hand motifs  178  
Talin αIIb; β  Structural cytoskeletal protein 179, 180  
α-Actinin  β  Structural cytoskeletal protein  181  
Skelemin  β  A myosin and intermediate filament-associated protein  182  
pp125FAK (focal adhesion kinase)  β  Protein tyrosine kinase localized to focal adhesions  183  
p59ILK (integrin-linked kinase) β  Contains ankyrin repeats and serine threonine kinase domain; overexpression inhibits cell adhesion and induces anchorage-independent growth  184  
Paxillin  β1 Adapter with SH2 and SH3 binding motifs and LIM domains  185 
ICAP-1  β1 only  Cell adhesion via β1 modulates phosphorylation state of ICAP-1  186 
Filamin  β2 Structural cytoskeletal protein 187  
Cytohesin-1  β2 only  Contains Sec7 and PH domains; guanine nucleotide exchange activity for ADP-ribosylation factor; overexpression increases αLβ2-mediated adhesion  121, 123 
β3-endonexin  β3 only Overexpression increases αIIbβ3affinity and adhesive function  118, 120  
p27BBP β4 May link β4 to the intermediate filament cytoskeleton in epithelial cells  188 
Rack 1 β1; β2; β5 Binds to the β tails via its 5-7th WD repeats in response to cell stimulation with phorbol ester 202 
Protein  Integrin Tail Partner  Notable Features Reference  
Calreticulin  α* Expression correlates with integrin-mediated cell adhesion; present in many subcellular locations.  173-176  
F-actin  α2 only Structural cytoskeletal protein  177  
Calcium- and integrin-binding protein (CIB)  αIIb only  Sequence homology to calcineurin B; contains 2 EF-hand motifs  178  
Talin αIIb; β  Structural cytoskeletal protein 179, 180  
α-Actinin  β  Structural cytoskeletal protein  181  
Skelemin  β  A myosin and intermediate filament-associated protein  182  
pp125FAK (focal adhesion kinase)  β  Protein tyrosine kinase localized to focal adhesions  183  
p59ILK (integrin-linked kinase) β  Contains ankyrin repeats and serine threonine kinase domain; overexpression inhibits cell adhesion and induces anchorage-independent growth  184  
Paxillin  β1 Adapter with SH2 and SH3 binding motifs and LIM domains  185 
ICAP-1  β1 only  Cell adhesion via β1 modulates phosphorylation state of ICAP-1  186 
Filamin  β2 Structural cytoskeletal protein 187  
Cytohesin-1  β2 only  Contains Sec7 and PH domains; guanine nucleotide exchange activity for ADP-ribosylation factor; overexpression increases αLβ2-mediated adhesion  121, 123 
β3-endonexin  β3 only Overexpression increases αIIbβ3affinity and adhesive function  118, 120  
p27BBP β4 May link β4 to the intermediate filament cytoskeleton in epithelial cells  188 
Rack 1 β1; β2; β5 Binds to the β tails via its 5-7th WD repeats in response to cell stimulation with phorbol ester 202 

*Unless specified otherwise, the integrin-binding protein has been shown to bind to more than type of α or β subunit.

Adapted and reprinted with permission.189 Reproduced from The Journal of Clinical Investigation, 1997, Vol. 100, pp. 1, by copyright permission of The American Society for Clinical Investigation.

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 αIIbβ3. CD47, also known as integrin-associated protein, spans the platelet plasma membrane five times and coimmunoprecipitates with β3integrins.128 So far, no direct role for CD47 in αIIbβ3 function has been demonstrated, either in platelets or in the CHO cell model system.129However, CD47 may function as a costimulatory agonist receptor in platelets because binding of thrombospondin to CD47 leads to activation of αIIbβ3 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 αIIbβ3 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 αIIbβ3 in platelet α-granule membranes and filopodia.133 Antibodies to CD9 can stimulate platelet aggregation in an Fc receptor-independent manner.134However, tetraspanins may exist in multimolecular complexes, and the interaction of CD9 with αIIbβ3 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 αIIbβ3 and other proteins remain a fertile area for further investigation.

Table 2.

Membrane Proteins Associated With Integrins

Protein  Associated Integrin  Reference  
CD47 (integrin-associated protein)  αIIbβ3; αVβ3; leukocyteresponse integrin 128, 190  
CD98  αIIbβ3 132 
Tetraspanins   133, 191-195  
 CD9 αIIbβ3; α3β1; α4β1; α5β1; α6β1 
 CD63 α3β1 
 CD81; NAG-2 α3β1; α6β1 
 CD151 α5β1 
EMMPRIN α3β1; α6β1 196  
Caveolin α1β1 135  
CD87 (urokinase plasminogen activator receptor)  αMβ2; αVβ3; αVβ5; α3β1; α5β1; α6β1 197-199  
CD16 (FcγRIIIB)  αMβ2 198 
Protein  Associated Integrin  Reference  
CD47 (integrin-associated protein)  αIIbβ3; αVβ3; leukocyteresponse integrin 128, 190  
CD98  αIIbβ3 132 
Tetraspanins   133, 191-195  
 CD9 αIIbβ3; α3β1; α4β1; α5β1; α6β1 
 CD63 α3β1 
 CD81; NAG-2 α3β1; α6β1 
 CD151 α5β1 
EMMPRIN α3β1; α6β1 196  
Caveolin α1β1 135  
CD87 (urokinase plasminogen activator receptor)  αMβ2; αVβ3; αVβ5; α3β1; α5β1; α6β1 197-199  
CD16 (FcγRIIIB)  αMβ2 198 

OUTSIDE-IN SIGNALING IN PLATELETS

Platelet functions regulated by outside-in signaling.

Signaling through αIIbβ3 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 αIIbβ3 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 αIIbβ3, 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 α-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).143-145Within 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 αIIbβ3 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 

Fig. 5.

Outside-in signaling through αIIbβ3 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).

Fig. 5.

Outside-in signaling through αIIbβ3 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).

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 β 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 αIIbβ3 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 αIIbβ3 provide strong evidence for involvement of the αIIb and β3cytoplasmic tails in outside-in signaling.107,108,156However, many fundamental questions remain. What is the role of tyrosine phosphorylation of the β3 tail in response to platelet aggregation?127 Can certain protein or lipid kinases and phosphatases couple directly to αIIbβ3? 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 αIIbβ3-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 αIIbβ3).162This allows facile categorization of the abnormality as either an αIIbβ3 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 αIIbβ3 function. For example, several dominant mutations introduced experimentally into the αIIb or β3 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 αIIbβ3 has expanded beyond the realm of the hematologist because of the development of pharmacological inhibitors of ligand binding to αIIbβ3 for prophylaxis and therapy of arterial thrombosis.169,170Abciximab, a chimeric mouse-human antibody that blocks ligand binding to αIIbβ3, is already licensed for use as adjunctive therapy in patients undergoing coronary angioplasty, and additional parenteral and orally active compounds are now in clinical trials. It is too early to predict the full range of indications for these agents or the degree of efficacy and risk of long-term use, but it is satisfying that platelet research has yielded the first integrin-based therapeutics. In this context, the orally active antiplatelet agents currently available in developed countries are, in one way or another, inhibitors of inside-out integrin signaling: aspirin inhibits cyclooxygenase-1 and, ultimately, the production of thromboxane A2; ticlopidine and clopidogrel inhibit signaling through the ADP receptor171; and phosphodiesterase inhibitors decrease catabolism of cyclic AMP, a suppressor of platelet activation. If the intracellular events responsible for αIIbβ3 signaling can be better defined, it may be possible to identify new integrin-proximal signaling proteins as drug targets.

ACKNOWLEDGMENT

The authors are grateful to our collaborators, past and present, and in particular Joan Brugge and Mark Ginsberg, for many of the concepts summarized herein, and to Mark Ginsberg and Martin Schwartz for critical review of the manuscript. Cited work from the authors' laboratory was supported by National Institutes of Health Grants No. HL56595 and HL57900 and from Cor Therapeutics, Inc.

Address reprint requests to Sanford J. Shattil, MD, Department of Vascular Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, VB-5, La Jolla, CA 92037.

REFERENCES

REFERENCES
1
Martin
P
Wound healing—Aiming for perfect skin regeneration.
Science
276
1997
75
2
Diacovo
TG
Roth
SJ
Buccola
JM
Bainton
DF
Springer
TA
Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta2-integrin CD11b/CD18.
Blood
88
1996
146
3
McEver
RP
Cummings
RD
Perspectives series: Cell adhesion in vascular biology. Role of PSGL-1 binding to selectins in leukocyte recruitment.
J Clin Invest
100
1997
485
4
Frenette
PS
Johnson
RC
Hynes
RO
Wagner
DD
Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin.
Proc Natl Acad Sci USA
92
1995
7450
5
Savage
B
Saldı́var
E
Ruggeri
ZM
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell
84
1996
289
6
Ruggeri ZM, FitzGerald GA, Shattil SJ: Platelet thrombus formation and anti-platelet therapy, in Chien KR, Breslow JL, Leiden JM, Rosenberg RD, Seidman C, Braunwald E (eds): Molecular Basis of Heart Disease. Philadelphia, PA, Saunders (in press)
7
Hynes
RO
Integrins: Versatility, modulation, and signaling in cell adhesion.
Cell
69
1992
11
8
Schwartz
MA
Schaller
MD
Ginsberg
MH
Integrins: Emerging paradigms of signal transduction.
Annu Rev Cell Biol
11
1995
549
9
Clark
EA
Brugge
JS
Integrins and signal transduction pathways. The road taken.
Science
268
1995
233
10
Sastry
SK
Horwitz
AF
Adhesion-growth factor interactions during differentiation: An integrated biological response.
Dev Biol
180
1996
455
11
Yamada
KM
Geiger
B
Molecular interactions in cell adhesion complexes.
Curr Opin Cell Biol
9
1997
76
12
Schlaepfer DD, Hunter T: Integrin signaling and tyrosine phosphorylation: Just the FAKs? Trends Cell Biol (in press)
13
Brass LF: Molecular basis for platelet activation, in Hoffman R, Benz E, Shattil S, Furie B, Cohen H, Silberstein L (eds): Hematology. Basic Principles and Practice. New York, NY, Churchill-Livingstone, 1995, p 1536
14
(suppl 4)
Fox
JEB
Platelet activation: New aspects.
Haemostasis
26
1996
102
15
Parise
LV
Phillips
DR
Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex.
J Biol Chem
260
1985
10698
16
Weisel
JW
Nagaswami
C
Vilaire
G
Bennett
JS
Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.
J Biol Chem
267
1992
16637
17
Plow
EF
D'Souza
SE
Ginsberg
MH
Ligand binding to GP IIb-IIIa: A status report.
Semin Thromb Hemost
18
1992
324
18
Bennett
JS
Vilaire
G
Exposure of platelet fibrinogen receptors by ADP and epinephrine.
J Clin Invest
64
1979
1393
19
Neri
D
Montigiani
S
Kirkham
PM
Biophysical methods for the determination of antigen-antibody affinites.
Trends Biochem Sci
14
1996
465
20
Miyamoto
S
Teramoto
H
Gutkind
JS
Yamada
KM
Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: Roles of integrin aggregation and occupancy of receptors.
J Cell Biol
135
1996
1633
21
Juliano
R
Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation.
Bioassays
18
1996
911
22
Peerschke
EI
Regulation of platelet aggregation by post-fibrinogen binding events.
Thromb Haemost
73
1995
862
23
Wagner
CL
Mascelli
MA
Neblock
DS
Weisman
HF
Coller
BS
Jordan
RE
Analysis of GP IIb/IIIa receptor number by quantification of 7E3 binding to human platelets.
Blood
88
1996
907
24
Frojmovic
M
Wong
T
Van de Ven
T
Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators.
Biophys J
59
1991
815
25
Nurden
AT
Association of fibrinogen-bound glycoprotein IIb-IIIa complexes on the activated platelet surface.
J Lab Clin Med
128
1996
7
26
Kleiman
NS
Raizner
AE
Jordan
R
Wang
AL
Norton
D
Mace
KF
Joshi
A
Coller
BS
Weisman
HF
Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: Implications for inhibition of the internal pool of GPIIb/IIIa receptors.
J Am Coll Cardiol
26
1995
1665
27
Woods
VL
Wolff
LE
Keller
DM
Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins.
J Biol Chem
261
1986
15242
28
Diamond
MS
Springer
TA
The dynamic regulation of integrin adhesiveness.
Curr Biol
4
1994
506
29
van Kooyk
Y
Figdor
CG
Signalling and adhesive properties of the integrin leucocyte function-associated antigen 1 (LFA-1).
Biochem Soc Trans
25
1997
515
30
Peerschke
EIB
Stabilization of platelet-fibrinogen interactions is an integral property of the glycoprotein IIb-IIIa complex.
J Lab Clin Med
124
1994
439
31
Müller
B
Zerwes
H-G
Tangemann
K
Peter
J
Engel
J
Two-step binding mechanism of fibrinogen to αIIbβ3 integrin reconstituted into planar lipid bilayers.
J Biol Chem
268
1993
6800
32
Simmons
SR
Albrecht
RM
Self-association of bound fibrinogen on platelet surfaces.
J Lab Clin Med
128
1996
39
33
Peerschke
EIB
Bound fibrinogen distribution on stimulated platelets—Examination by confocal scanning laser microscopy.
Am J Pathol
147
1995
678
34
Fox
J
Shattil
SJ
Kinlough-Rathbone
R
Richardson
M
Packham
MA
Sanan
DA
The platelet cytoskeleton stabilizes the interaction between αIIbβ3 and its ligand and induces selective movements of ligand-occupied integrin.
J Biol Chem
271
1996
7004
35
Kucik
DF
Dustin
ML
Miller
JM
Brown
EJ
Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes.
J Clin Invest
97
1996
2139
36
Wencel-Drake
JD
Boudignon-Proudhon
C
Dieter
MG
Criss
AB
Parise
LV
Internalization of bound fibrinogen modulates platelet aggregation.
Blood
87
1996
602
37
Leung
L
Nachman
R
Molecular mechanisms of platelet aggregation.
Annu Rev Med
37
1986
179
38
Abrams
C
Deng
J
Steiner
B
Shattil
SJ
Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to the activated form of integrin αIIbβ3.
J Biol Chem
269
1994
18781
39
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
266
1991
7345
40
Du
X
Gu
M
Weisel
J
Nagaswami
C
Bennett
JS
Bowditch
R
Ginsberg
MH
Long range propagation of conformational changes in integrin αIIbβ3.
J Biol Chem
268
1993
23087
41
Springer
TA
Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain.
Proc Natl Acad Sci USA
94
1997
65
42
Tozer
EC
Liddington
RC
Sutcliffe
MJ
Smeeton
AH
Loftus
JC
Ligand binding to integrin αIIbβ3 is dependent on a MIDAS-like domain in the β3 subunit.
J Biol Chem
271
1996
21978
43
Lee
JO
Riev
P
Aranout
MA
Liddington
R
Crystal structure of the A-domain from the A-subunit of integrin CR3 (CD11B/CD18).
Cell
80
1995
631
44
Qu
AD
Leahy
DJ
The role of divalent cation in the structure of the I-domain from the CD11a/CD18 integrin.
Structure
4
1996
931
45
Loftus
JC
Liddington
RC
Cell adhesion in vascular biology. New insights into integrin-ligand interaction.
J Clin Invest
99
1997
2302
46
Farrell
DH
Thiagarajan
P
Chung
DW
Davie
EW
Role of fibrinogen α and γ chain sites in platelet aggregation.
Proc Natl Acad Sci USA
89
1992
10729
47
Rooney
MM
Parise
LV
Lord
ST
Dissecting clot retraction and platelet aggregation—Clot retraction does not require an intact fibrinogen gamma chain.
J Biol Chem
271
1996
8553
48
Ugarova
TP
Budzynski
AZ
Shattil
SJ
Ruggeri
ZM
Ginsberg
MH
Plow
EF
Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa.
J Biol Chem
268
1993
21080
49
Savage
B
Bottini
E
Ruggeri
ZM
Interaction of integrin αIIbβ3 with multiple fibrinogen domains during platelet adhesion.
J Biol Chem
270
1995
28812
50
Hung
DT
Vu
T-KH
Wheaton
VI
Ishii
K
Coughlin
SR
Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.
J Clin Invest
89
1992
1350
51
Brass
LF
Manning
DR
Cichowski
K
Abrams
CS
Signaling through G proteins in platelets: To the integrins and beyond.
Thromb Haemost
78
1997
581
52
Gachet
C
Hechler
B
Léon
C
Vial
C
Leray
C
Ohlmann
P
Cazenave
JP
Activation of ADP receptors and platelet function.
Thromb Haemost
78
1997
271
53
Offermanns
S
Toombs
CF
Hu
YH
Simon
MI
Defective platelet activation in Gαq-deficient mice.
Nature
389
1997
183
54
Jackson
SP
Schoenwaelder
SM
Yuan
YP
Salem
HH
Cooray
P
Non-receptor protein tyrosine kinases and phosphatases in human platelets.
Thromb Haemost
76
1996
640
55
Raja
S
Avraham
S
Avraham
H
Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism.
J Biol Chem
272
1997
10941
56
Tate
BF
Rittenhouse
SE
Thrombin activation of human platelets causes tyrosine phosphorylation of PLC-gamma2.
Biochim Biophys Acta Mol Cell Res
1178
1993
281
57
Cichowski
K
Brugge
JS
Brass
LF
Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95vav, in platelets.
J Biol Chem
271
1996
7544
58
Rosa
JP
Artcanuthurry
V
Grelac
F
Maclouf
J
Caen
JP
Lévy-Toledano
S
Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: Evidence that phosphorylation of cortactin and a 64-kD protein is dependent on thrombin activation and integrin αIIbβ3.
Blood
89
1997
4385
59
Lerea
KM
Tonks
NK
Krebs
EG
Fischer
EH
Glomset
JA
Vanadate and molybdate increase tyrosine phosphorylation in a 50-kilodalton protein and stimulate secretion in electropermeabilized platelets.
Biochemistry
28
1989
9286
60
(abstr, suppl 1)
Law
DA
Nannizzi-Alaimo
L
Ministri
K
Hughes
P
Turner
M
Shattil
SJ
Ginsberg
MH
Tybulewicz
V
Phillips
DR
Syk-deficient platelets demonstrate a role for Syk in αIIbβ3 inside-out signaling and highlight the lack of specificity of the tyrosine kinase inhibitor, piceatannol.
Blood
90
1997
425a
61
Huang
M-M
Indik
Z
Brass
LF
Hoxie
JA
Schrieber
AD
Brugge
JS
Activation of FcγRII induces tyrosine phosphorylation of multiple proteins including FcγRII.
J Biol Chem
267
1992
5467
62
Chacko
GW
Brandt
JT
Coggeshall
KM
Anderson
CL
Phosphoinositide 3-kinase and p72syk noncovalently associate with the low affinity Fcgamma receptor on human platelets through an immunoreceptor tyrosine-based activation motif—Reconstitution with synthetic phosphopeptides.
J Biol Chem
271
1996
10775
63
Ruggeri
ZM
Mechanisms initiating platelet thrombus formation.
Thromb Haemost
78
1997
611
64
Rand
JH
Glanville
RW
Wu
XX
Ross
JM
Zangari
M
Gordon
RE
Schwartz
E
Potter
BJ
The significance of subendothelial von Willebrand factor.
Thromb Haemost
78
1997
445
65
Santoro
SA
Zutter
MM
The α2β1 integrin: A collagen receptor on platelets and other cells.
Thromb Haemost
74
1995
813
66
Diaz-Ricart
M
Tandon
NN
Carretero
M
Ordinas
A
Bastida
E
Jamieson
GA
Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood.
Blood
82
1993
491
67
Moroi
M
Jung
SM
Platelet receptors for collagen.
Thromb Haemost
78
1997
439
68
Tsuji
M
Ezumi
Y
Arai
M
Takayama
H
A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets.
J Biol Chem
272
1997
23528
69
Gibbins
JM
Okuma
M
Farndale
R
Barnes
M
Watson
SP
Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.
FEBS Lett
413
1997
255
70
Gibbins
J
Asselin
J
Farndale
R
Barnes
M
Law
CL
Watson
SP
Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.
J Biol Chem
271
1996
18095
71
Asselin
J
Gibbins
JM
Achison
M
Lee
YH
Morton
LF
Farndale
RW
Barnes
MJ
Watson
SP
Collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase Cgamma2 in platelets independent of the integrin α2β1.
Blood
89
1997
1235
72
Polgar
J
Clemetson
JM
Kehrel
BE
Wiedemann
M
Magnenat
EM
Wells
TNC
Clemetson
KJ
Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.
J Biol Chem
272
1997
13576
73
Poole
A
Gibbins
JM
Turner
M
van Vugt
MJ
van den Winkel
JGJ
Saito
T
Tybulewicz
VLJ
Watson
SP
The Fc receptor γ-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.
EMBO J
16
1997
2333
74
Ozaki
Y
Satoh
K
Yatomi
Y
Miura
S
Fujimura
Y
Kume
S
Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor.
Biochim Biophys Acta
1243
1995
482
75
Shattil
SJ
Brass
LF
Induction of the fibrinogen receptor on human platelets by intracellular mediators.
J Biol Chem
262
1987
992
76
Shiraga
M
Tomiyama
Y
Honda
S
Kashiwagi
H
Kosugi
S
Handa
M
Ikeda
Y
Kanakura
Y
Kurata
Y
Matsuzawa
Y
Affinity modulation of the platelet integrin alpha IIb beta 3 by alpha-chymotrypsin: A possible role for Na+/Ca2+ exchanger.
Blood
88
1996
2594
77
Shattil
SJ
Cunningham
M
Wiedmer
T
Zhao
J
Sims
PJ
Brass
LF
Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.
J Biol Chem
267
1992
18424
78
Hemmings
BA
Update: Signal transduction—PH domains—A universal membrane adapter.
Science
275
1997
1899
79
Li
JX
Zhu
ZX
Bao
ZH
Role of MacMARCKS in integrin-dependent macrophage spreading and tyrosine phosphorylation of paxillin.
J Biol Chem
271
1996
12985
80
Rittenhouse
SE
Phosphoinositide 3-kinase activation and platelet function.
Blood
88
1996
4401
81
Shimizu
Y
Hunt SW
III
Regulating integrin-mediated adhesion: One more function for PI 3-kinase?
Immunol Today
17
1996
565
82
Tang
XW
Downes
CP
Purification and characterization of Gβgamma-responsive phosphoinositide 3-kinases from pig platelet cytosol.
J Biol Chem
272
1997
14193
83
Gutkind
JS
Lacal
PM
Robbins
KC
Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.
Mol Cell Biol
10
1990
3806
84
Yanagi
S
Sada
K
Tohyama
Y
Tsubokawa
M
Nagai
K
Yonezawa
K
Yamamura
H
Translocation, activation and association of protein-tyrosine kinase (p72syk) with phosphatidylinositol 3-kinase are early events during platelet activation.
Eur J Biochem
224
1994
329
85
Toker
A
Cantley
LC
Signaling through the lipid products of phosphoinositides.
Nature
387
1997
673
86
(abstr, suppl 1)
Kovacsovics
TJ
Hartwig
JH
Cantley
LC
Toker
A
Irreversible platelet aggregation and prolonged pleckstrin phosphorylation are mediated by the lipid products of phosphphatidylinositol 3-kinase.
Blood
86
1995
454a
87
Hartwig
JH
Kung
S
Kovacsovics
T
Janmey
PA
Cantley
LC
Stossel
TP
Toker
A
D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.
J Biol Chem
271
1996
32986
88
Toker
A
Bachelot
C
Chen
CS
Falck
JR
Hartwig
JH
Cantley
LC
Kovacsovics
TJ
Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.
J Biol Chem
270
1995
29525
89
Hartwig
JH
Bokoch
GM
Carpenter
CL
Janmey
PA
Taylor
LA
Toker
A
Stossel
TP
Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets.
Cell
82
1995
643
90
Morii N, Teru-uchi T, Tominaga T, Kumagai N, Kozaki S, Ushikubi F, Narumiya S: A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem 267:20921, 1992
91
Leng L, Kashiwagi H, Ren X-D, Shattil SJ: Rho A and the function of platelet integrin αIIbβ3. Blood (in press)
92
Machesky
LM
Hall
A
Rho: A connection between membrane receptor signalling and the cytoskeleton.
Trends Cell Biol
6
1996
304
93
Zhang
Z
Vuori
K
Wang
H-G
Reed
JC
Ruoshlati
E
Integrin activation by R-ras.
Cell
85
1996
61
94
Shock
DD
He
K
Wencel-Drake
JD
Parise
LV
Ras activation in platelets following stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.
Biochem J
321
1997
525
95
Franke
B
Akkerman
JWN
Bos
JL
Rapid Ca2+-mediated activation of Rap1 in human platelets.
EMBO J
16
1997
252
96
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
14
1994
463
97
Hughes
PE
Renshaw
MW
Pfaff
M
Forsyth
J
Keivens
VM
Schwartz
MA
Ginsberg
MH
Suppression of integrin activation: A novel function of a Ras/Raf-initiated MAP-kinase pathway.
Cell
88
1996
521
98
Nadal
F
Levy-Toledano
S
Grelac
F
Caen
JP
Rosa
JP
Bryckaert
M
Negative regulation of mitogen-activated protein kinase activation by integrin αIIbβ3 in platelets.
J Biol Chem
272
1997
22381
99
Freedman
JE
Loscalzo
J
Barnard
MR
Alpert
C
Keaney JF Jr
Michelson
AD
Nitric oxide released from activated platelets inhibits platelet recruitment.
J Clin Invest
100
1997
350
100
Freedman
JE
Loscalzo
J
Benoit
SE
Valeri
R
Barnard
MR
Michelson
AD
Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.
J Clin Invest
97
1996
979
101
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
14
1995
19
102
Eigenthaler
M
Walter
U
Signal transduction and cyclic nucleotides in human platelets.
Thromb Haemorrh Disorders
8
1994
41
103
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
273
1991
115
104
Marcus
AJ
Broekman
MJ
Drosopoulos
JHF
Islam
N
Alyonycheva
TN
Safier
LB
Hajjar
KA
Posnett
DN
Schoenborn
MA
Schooley
KA
Gayle
RB
Maliszewski
CR
The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.
J Clin Invest
99
1997
1351
105
Vassbotn
FS
Havnen
OK
Heldin
C-H
Holmsen
H
Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor α-receptor.
J Biol Chem
269
1994
13874
106
Chen
Y-P
Djaffar
I
Pidard
D
Steiner
B
Cieutat
A-M
Caen
JP
Rosa
J-P
Ser-752 → Pro mutation in the cytoplasmic domain of integrin β3 subunit and defective activation of platelet integrin αIIb β3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.
Proc Natl Acad Sci USA
89
1992
10169
107
Wang
R
Shattil
SJ
Ambruso
DR
Newman
PJ
Truncation of the cytoplasmic domain of β3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin αIIbβ3 complex.
J Clin Invest
100
1997
2393
108
Chen
Y-P
O'Toole
TE
Ylänne
J
Rosa
J-P
Ginsberg
MH
A point mutation in the integrin β3 cytoplasmic domain (S752→P) impairs bidirectional signaling through αIIb β3 (platelet glycoprotein IIb-IIIa).
Blood
84
1994
1857
109
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 signaling.
J Cell Biol
124
1994
1047
110
O'Toole
TE
Ylanne
J
Culley
BM
Regulation of integrin affinity states through an NPXY motif in the β subunit cytoplasmic domain.
J Biol Chem
270
1995
8553
111
Hughes
PE
O'Toole
TE
Ylanne
J
Shattil
SJ
Ginsberg
MH
The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand-binding affinity.
J Biol Chem
270
1995
12411
112
Hughes
PE
Diaz-Gonzalez
F
Leong
L
Wu
CY
McDonald
JA
Shattil
SJ
Ginsberg
MH
Breaking the integrin hinge—A defined structural constraint regulates integrin signaling.
J Biol Chem
271
1996
6571
113
Loh
E
Qi
WW
Vilaire
G
Bennett
JS
Effect of cytoplasmic domain mutations on the agonist-stimulated ligand binding activity of the platelet integrin αIIbβ3.
J Biol Chem
271
1996
30233
114
Chen
Y-P
O'Toole
TE
Shipley
T
Forsyth
J
LaFlamme
SE
Yamada
KM
Shattil
SJ
Ginsberg
MH
“Inside-out” signal transduction inhibited by isolated integrin cytoplasmic domains.
J Biol Chem
269
1994
18307
115
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
93
1996
11819
116
Muir
TW
Williams
MJ
Ginsberg
MH
Kent
SBH
Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: Integrin αIIbβ3 (platelet GPIIb-IIIa).
Biochemistry
33
1994
7701
117
Haas
TA
Plow
EF
The cytoplasmic domain of αIIbβ3. A ternary complex of the integrin alpha and beta subunits and a divalent cation.
J Biol Chem
271
1996
6017
118
Shattil
SJ
O'Toole
T
Eigenthaler
M
Thon
V
Williams
M
Babior
BM
Ginsberg
MH
β3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin β3 subunit.
J Cell Biol
131
1995
807
119
Eigenthaler
M
Hofferer
L
Shattil
SJ
Ginsberg
MH
A conserved sequence motif in the integrin β3 cytoplasmic domain is required for its specific interaction with β3-endonexin.
J Biol Chem
272
1997
7693
120
Kashiwagi
H
Schwartz
MA
Eigenthaler
MA
Davis
KA
Ginsberg
MH
Shattil
SJ
Affinity modulation of platelet integrin αIIbβ3 by β3-endonexin, a selective binding partner of the β3 integrin cytoplasmic tail.
J Cell Biol
137
1997
1433
121
Kolanus
W
Nagel
W
Schiller
B
Zeitlmann
L
Godar
S
Stockinger
H
Seed
B
Alpha-L-Beta-2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule.
Cell
86
1996
233
122
Klarlund
JK
Guilherme
A
Holik
JJ
Virbasius
JV
Chawla
A
Czech
MP
Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains.
Science
275
1997
1927
123
Meacci
E
Tsai
SC
Adamik
R
Moss
J
Vaughn
M
Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor.
Proc Natl Acad Sci USA
94
1997
1745
124
Radeva
G
Petrocelli
T
Behrend
E
Leung-Hagesteijn
C
Filmus
J
Slingerland
J
Dedhar
S
Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.
J Biol Chem
272
1997
13937
125
Hillery
CA
Smyth
SS
Parise
LV
Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.
J Biol Chem
266
1991
14663
126
Van Willigen
G
Hers
I
Gorter
G
Akkerman
J-WN
Exposure of ligand-binding sites on platelet integrin αIIb/β3 by phosphorylation of the β3 subunit.
Biochem J
314
1996
769
127
Law
DA
Nannizzi-Alaimo
L
Phillips
DR
Outside-in signal transduction: αIIbβ3 (GP IIb-IIIa) tyrosine phosphorylation induced by platelet aggregation.
J Biol Chem
271
1996
10811
128
Lindberg
FP
Lublin
DM
Telen
MJ
Veile
RA
Miller
YE
Donis-Keller
H
Brown
EJ
Rh-related antigen CD47 is the signal-transducer integrin-associated protein.
J Biol Chem
269
1994
1567
129
Fujimoto
T
Fujimura
K
Noda
M
Takafuta
T
Shimomura
T
Kuramoto
A
50-kD integrin-associated protein does not detectably influence several functions of glycoprotein IIb-IIIa complex in human platelets.
Blood
86
1995
2174
130
Chung
J
Gao
AG
Frazier
WA
Thrombospondin acts via integrin-associated protein to activate the platelet integrin αIIbβ3.
J Biol Chem
272
1997
14740
131
Dorahy
DJ
Thorne
RF
Fecondo
JV
Burns
GF
Stimulation of platelet activation and aggregation by a carboxy-terminal peptide from thrombospondin binding to the integrin-associated protein receptor.
J Biol Chem
272
1997
1323
132
Fenczik
CA
Ramos
JW
Ginsberg
MH
Complementation of dominant suppression implicates CD98 in integrin activation.
Nature
390
1997
81
133
Brisson
C
Azorsa
DO
Jennings
LK
Moog
S
Cazenave
JP
Lanza
F
Co-localization of CD9 and GPIIb-IIIa (αIIb β3 integrin) on activated platelet pseudopods and α-granule membranes.
Histochem J
29
1997
153
134
Slupsky
JR
Kamiguti
AS
Rhodes
NP
Cawley
JC
Shaw
ARE
Zuzel
M
The platelet antigens CD9, CD42 and integrin αIIb βIIIa can be topographically associated and transduce functionally similar signals.
Eur J Biochem
244
1997
168
135
Wary
KK
Mainiero
F
Isakoff
SJ
Marcantonio
EE
Giancotti
FG
The adaptor protein Shc couples a class of integrins to the control of cell cycle progression.
Cell
87
1996
733
136
Dorahy
DJ
Berndt
MC
Burns
GF
Capture by chemical crosslinkers provides evidence that integrin αIIbβ3 forms a complex with protein tyrosine kinases in intact platelets.
Biochem J
309
1995
481
137
Savage
B
Shattil
SJ
Ruggeri
ZM
Modulation of platelet function through adhesion receptors. A dual role for glycoprotein IIb-IIIa (integrin αIIbβ3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.
J Biol Chem
267
1992
11300
138
Cohen
I
Gerrard
JM
White
JG
Ultrastructure of clots during isometric contraction.
J Cell Biol
93
1982
775
139
Chen
Y-P
O'Toole
TE
Leong
L
Liu
B-Q
Diaz-Gonzalez
F
Ginsberg
MH
β3 integrin-mediated fibrin clot retraction by nucleated cells: Differing behavior of αIIbβ3 and αvβ3.
Blood
86
1995
2606
140
Schoenwaelder
SM
Yuan
YP
Cooray
P
Salem
HH
Jackson
SP
Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin αIIbβ3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots.
J Biol Chem
272
1997
1694
141
Fox
JEB
Reynolds
CC
Austin
CD
The role of calpain in stimulus-response coupling: Evidence that calpain mediates agonist-induced expression of procoagulant activity in platelets.
Blood
76
1990
2510
142
Narumiya S: The small GTPase Rho: Cellular functions and signal transduction. J Biochem (Tokyo) 120:215, 1996
143
Hartwig
JH
Mechanisms of actin rearrangements mediating platelet activation.
J Cell Biol
118
1992
1421
144
Fox
JEB
The platelet cytoskeleton.
Thromb Haemost
70
1993
884
145
Shattil
SJ
Ginsberg
MH
Brugge
JS
Adhesive signaling in platelets.
Curr Opin Cell Biol
6
1994
695
146
Yuan
YP
Dopheide
SM
Ivanidis
C
Salem
HH
Jackson
SP
Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets—Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin αIIbβ3) in von Willebrand factor-induced signal transduction.
J Biol Chem
272
1997
21847
147
Gao
J
Zoller
K
Ginsberg
MH
Brugge
JS
Shattil
SJ
Regulation of the pp72Syk protein tyrosine kinase by platelet integrin αIIbβ3.
EMBO J
16
1997
6414
148
Nachmias
VT
Golla
R
Vinculin in relation to stress fibers in spread platelets.
Cell Motil Cytoskeleton
20
1991
190
149
Laffargue
M
Monnereau
L
Tuech
J
Ragab
A
Ragab-Thomas
J
Payrastre
B
Raynal
P
Chap
H
Integrin-dependent tyrosine phosphorylation and cytoskeletal translocation of Tec in thrombin-activated platelets.
Biochem Biophys Res Commun
238
1997
247
150
Frangione
JV
Oda
A
Smith
M
Salzman
EW
Neel
BG
Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets.
EMBO J
12
1993
4843
151
Ezumi
Y
Takayama
H
Okuma
M
Differential regulation of protein-tyrosine phosphatases by integrin αIIbβ3 through cytoskeletal reorganization and tyrosine phosphorylation in human platelets.
J Biol Chem
270
1995
11927
152
Li
RY
Ragab
A
Gaits
F
Ragab-Thomas
JMF
Chap
H
Thrombin-induced redistribution of protein-tyrosine-phosphatases to the cytoskeletal complexes in human platelets.
Cell Mol Biol
40
1994
665
153
Parsons
JT
Integrin-mediated signalling: Regulation by protein tyrosine kinases and small GTP-binding proteins.
Curr Opin Cell Biol
8
1996
146
154
Ilic
D
Furuta
Y
Kanazawa
S
Takeda
N
Sobue
K
Nakatsuji
N
Nomura
S
Fujimoto
J
Okada
M
Yamamoto
T
Aizawa
S
Reduced cell motility and enhanced focal adhesion formation in cells from FAK-deficient mice.
Nature
377
1995
539
155
Shattil
SJ
Haimovich
B
Cunningham
M
Lipfert
L
Parsons
JT
Ginsberg
MH
Brugge
JS
Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors.
J Biol Chem
269
1994
14738
156
Leong
L
Hughes
PE
Schwartz
MA
Ginsberg
MH
Shattil
SJ
Integrin signaling: Roles for the cytoplasmic tails of αIIbβ3 in the tyrosine phosphorylation of pp125FAK.
J Cell Sci
108
1995
3817
157
Guinebault
C
Payrastre
B
Racaud-Sultan
C
Mazarguil
H
Breton
M
Mauco
G
Plantavid
M
Chap
H
Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85-α with actin filaments and focal adhesion kinase.
J Cell Biol
129
1995
831
158
Hinchliffe
KA
Irvine
RF
Divecha
N
Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.
EMBO J
15
1996
6516
159
Toyoda
H
Nakai
K
Omay
SB
Shima
H
Nagao
M
Shiku
H
Nishikawa
M
Differential association of protein Ser/Thr phosphatase types 1 and 2A with the cytoskeleton upon platelet activation.
Thromb Haemost
76
1996
1053
160
Dash
D
Aepfelbacher
M
Siess
W
Integrin αIIbβ3-mediated translocation of CDC42Hs to the cytoskeleton in stimulated human platelets.
J Biol Chem
270
1995
17321
161
Gironcel
D
Racaud-Sultan
C
Payrastre
B
Haricot
M
Borchert
G
Kieffer
N
Breton
M
Chap
H
αIIbβ3-integrin mediated adhesion of human platelets to a fibrinogen matrix triggers phospholipase C activation and phosphatidylinositol 3′,4′-bisphosphate accumulation.
FEBS Lett
389
1996
253
162
Michelson
AD
Flow cytometry: A clinical test of platelet function.
Blood
87
1996
4925
163
Ginsberg
MH
Frelinger
AL
Lam
SC-T
Forsyth
J
McMillan
R
Plow
EF
Shattil
SJ
Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies.
Blood
76
1990
2017
164
Gabbeta
J
Yang
X
Sun
L
McLane
MA
Niewiarowski
S
Rao
AK
Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation.
Blood
87
1996
1368
165
Gabbeta
J
Yang
X
Kowalska
MA
Sun
L
Dhanasekaran
N
Rao
AK
Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses.
Proc Natl Acad Sci USA
94
1997
8750
166
Nurden
P
Savi
P
Heilmann
E
Bihour
C
Herbert
J-M
Maffrand
J-P
Nurden
A
An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.
J Clin Invest
95
1995
1612
167
Lages
B
Shattil
SJ
Bainton
DF
Weiss
HJ
Decreased content and surface expression of α-granule membrane protein GMP-140 in one of two types of platelet γδ storage pool deficiency.
J Clin Invest
87
1991
919
168
Lages
B
Sussman
II
Levine
SP
Coletti
D
Weiss
HJ
Platelet alpha granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation in a new patient with gray platelet syndrome (α-storage pool deficiency).
J Lab Clin Med
129
1997
364
169
Lefkovits
J
Plow
EF
Topol
EJ
Mechanisms of disease: Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine.
N Engl J Med
332
1995
1553
170
Coller
BS
Platelet GPIIb/IIIa antagonists: The first anti-integrin receptor therapeutics.
J Clin Invest
99
1997
1467
171
(suppl 4)
Nurden
AT
New thoughts on strategies for modulating platelet function through the inhibition of surface receptors.
Haemostasis
26
1996
78
172
Chothia
C
Jones
EY
The molecular structure of cell adhesion molecules.
Annu Rev Biochem
66
1997
823
173
Coppolino
M
Leung-Hagesteijn
C
Dedhar
S
Wilkins
J
Inducible interaction of integrin α2β1 with calreticulin—Dependence on the activation state of the integrin.
J Biol Chem
270
1995
23132
174
Opas
M
Szewczenko-Pawlikowski
M
Jass
GJ
Mesaeli
N
Michalak
M
Calreticulin modulates cell adhesiveness via regulation of vinculin expression.
J Cell Biol
135
1996
1913
175
Zhu
Q
Zelinka
P
White
T
Tanzer
ML
Calreticulin-integrin bidirectional signaling complex.
Biochem Biophys Res Commun
232
1997
354
176
Coppolino
MG
Woodside
MJ
Demaurex
N
Grinstein
S
St-Arnaud
R
Dedhar
S
Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion.
Nature
386
1997
843
177
Kieffer
JD
Plopper
G
Ingber
DE
Hartwig
JH
Kupper
TS
Direct binding of F actin to the cytoplasmic domain of the α2 integrin chain in vitro.
Biochem Biophys Res Commun
217
1995
466
178
Naik
UP
Patel
PM
Parise
LV
Identification of a novel calcium binding protein that interacts with the integrin αIIb cytoplasmic domain.
J Biol Chem
272
1997
4651
179
Horwitz
A
Duggan
K
Buck
C
Beckerle
MC
Burridge
K
Interaction of plasma membrane fibronectin receptor with talin—A transmembrane linkage.
Nature
320
1986
531
180
Knezevic
I
Leisner
TM
Lam
SCT
Direct binding of the platelet integrin αIIbβ3 (GPIIb-IIIa) to talin—Evidence that interaction is mediated through the cytoplasmic domains of both αIIb and β3.
J Biol Chem
271
1996
16416
181
Otey
CA
Vasquez
GB
Burridge
K
Erickson
BW
Mapping of the α-actinin binding site within the β1 integrin cytoplasmic domain.
J Biol Chem
268
1993
21193
182
Reddy KB, Gascard P, Price MG, Fox JE: Identification and characterization of a specific interaction between skelemin and beta integrin cytoplasmic tails. Circ 94:I-98, 1996 (abstr)
183
Schaller
MD
Otey
CA
Hildebrand
JD
Parsons
JT
Focal adhesion kinase and paxillin bind to peptides mimicking β integrin cytoplasmic domains.
J Cell Biol
130
1995
1181
184
Hannigan
GE
Leung-Hagesteijn
C
Fitz-Gibbon
L
Coppolino
MG
Radeva
G
Filmus
J
Bell
JC
Dedhar
S
Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.
Nature
379
1996
91
185
Tanaka
T
Yamaguchi
R
Sabe
H
Sekiguchi
K
Healy
JM
Paxillin association in-vitro with integrin cytoplasmic domain peptides.
FEBS Lett
399
1996
53
186
Chang
DD
Wong
C
Smith
H
Liu
J
ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.
J Cell Biol
138
1997
1149
187
Sharma
CP
Ezzell
RM
Arnaout
MA
Direct interaction of filamin (ABP-280) with the β2-integrin subunit CD18.
J Immunol
154
1995
3461
188
Biffo
S
Sanvito
F
Costa
S
Preve
L
Pignatelli
R
Spinardi
L
Marchisio
PC
Isolation of a novel beta4 integrin-binding protein (p27(BBP)) highly expressed in epithelial cells.
J Biol Chem
272
1997
30314
189
Shattil
SJ
Ginsberg
MH
Integrin signaling in vascular biology.
J Clin Invest
100
1997
1
190
(abstr)
Lindberg
F
Brown
EJ
Cloning and expression of the integrin associated protein (IAP), an integral membrane protein involved in integrin signaling.
Mol Biol Cell
3
1992
95a
191
Berditchevski
F
Bazzoni
F
Hemler
ME
Specific association of CD63 with the VLA-3 and VLA-6 integrins.
J Biol Chem
270
1995
17784
192
Berditchevski
F
Zutter
MM
Hemler
ME
Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).
Mol Biol Cell
7
1996
193
193
Sincock
PM
Mayrhofer
G
Ashman
LK
Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: Comparison with CD9, CD63, and alpha5beta1 integrin.
J Histochem Cytochem
45
1997
515
194
Maecker HT, Todd SC, Levy S: The tetraspanin superfamily: Molecular facilitators. FASEB J 11:428, 1997
195
Tachibana
I
Bodorova
J
Berditchevski
F
Zutter
MM
Hemler
ME
NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins.
J Biol Chem
272
1997
29181
196
Berditchevski
F
Chang
S
Bodorova
J
Hemler
ME
Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with emmprin/basigin/ox47/m6.
J Biol Chem
272
1997
29174
197
Wei
Y
Lukashev
M
Simon
DI
Bodary
SC
Rosenberg
S
Doyle
MV
Chapman
HA
Regulation of integrin function by the urokinase receptor.
Science
273
1996
1551
198
Xue
W
Kindzelskii
AL
Todd
RF
Petty
HR
Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes.
J Immunol
152
1994
4630
199
Xue
W
Mizukami
I
Todd RF
III
Petty
HR
Urokinase-type plasminogen activator receptors associate with β1 and β3 integrins of fibrosarcoma cells: Dependence on extracellular matrix components.
Cancer Res
57
1997
1682
200
Giurato
S
Payrastre
B
Drayer
AL
Plantavid
M
Woscholski
R
Parker
P
Erneux
C
Chap
H
Tyrosine phosphorylation and relocation of SHIP are integrin-mediated in the thrombin-stimulated human platelets.
J Biol Chem
272
1997
26857
201
Fujita
A
Saito
Y
Ishizaki
T
Maekawa
M
Fujisawa
K
Ushikabi
F
Narumiya
S
Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets.
Biochem J
328
1997
769
202
Lilienthal
J
Chang
DD
Rak1, a receptor for activated protein kinase C, interacts with integrin β subunit.
J Biol Chem
273
1998
2379