The major platelet integrin, αIIbβ3, is required for platelet interactions with proteins in plasma and the extracellular matrices (ECMs) that are essential for platelet adhesion and aggregation during hemo stasis and arterial thrombosis. Lig and binding to αIIbβ3 is controlled by inside-out signals that modulate receptor conformation and clustering. In turn, ligand binding triggers outside-in signals through αIIbβ3 that, when disrupted, can cause a bleeding diathesis. In the past 5 years there has been an explosion of knowledge about the structure and function ofαIIbβ3 and the related integrin, αVβ3. These developments are discussed here, and current models of bidirectional αIIbβ3 signaling are presented as frameworks for future investigations. An understanding that αIIbβ3 functions as a dynamic molecular scaffold for extracellular and intracellular proteins has translated into diagnostic and therapeutic insights relevant to hematology and cardiovascular medicine, and further advances can be anticipated. (Blood. 2004;104:1606-1615)

The platelet is a tightly regulated adhesion machine. Restrained in its functions while in the bloodstream, its adhesive, hemostatic, and proinflammatory capabilities are unleashed at sites of vessel injury to generate the primary hemostatic plug, catalyze fibrin formation, and supply soluble and membrane-bound factors that promote wound healing.1  While platelets can adhere to damaged endothelial cells,2  their principle adhesive surface is the extracellular matrix (ECM), which becomes exposed in injured vessels and offers a panoply of ligands for platelet adhesion receptors.3  Within this context, integrin adhesion receptors, and αIIbβ3 in particular, play critical roles in platelet function.

Integrins are heterodimeric (αβ) type I transmembrane receptors, each subunit typically containing a relatively large extracellular domain, a single-pass transmembrane domain, and a short cytoplasmic tail composed of 20 to 60 amino acids.4  Platelets express several integrins (αIIbβ3, also called glycoprotein IIb-IIIa [GPIIb-IIIa]; αVβ3; α2β1; α5β1; α6β1). Integrins are, in effect, “2-faced” receptors, one face oriented to the extracellular space and interactive with cognate ECM ligands and the other oriented to the cell interior and interactive with cytoplasmic proteins. Ligand binding to either face can trigger information transfer, or signaling, across the plasma membrane to “activate” cellular functions at the other face. Figure 1 illustrates this bidirectional signaling using αIIbβ3 as an example.

Figure 1.

Integrin activation is bidirectional and reciprocal. The αIIbβ3 equilibrates between resting and activated states, the resting state predominating in unstimulated platelets and the activated state in stimulated platelets. Conversion from resting to activated does not imply a single, abrupt change but rather a series of coordinated and linked conformational transitions. (A) Inside-out signaling. Agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails (in this case the β3 tail). This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, von Willebrand factor (VWF), and fibronectin. Affinity modulation can be monitored in living cells with engineered monovalent Fab fragments derived from ligand-mimetic monoclonal antibodies.35,50,137  Plasma fibrinogen and VWF support platelet aggregation at low and high shear rates, respectively, by bridging αIIbβ3 receptors on adjacent platelets.3  Studies in mice deficient in fibrinogen and VWF indicate that plasma fibronectin can also promote thrombus initiation, growth, and stability at high shear rates.138  (B) Outside-in signaling. Extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tails. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.

Figure 1.

Integrin activation is bidirectional and reciprocal. The αIIbβ3 equilibrates between resting and activated states, the resting state predominating in unstimulated platelets and the activated state in stimulated platelets. Conversion from resting to activated does not imply a single, abrupt change but rather a series of coordinated and linked conformational transitions. (A) Inside-out signaling. Agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails (in this case the β3 tail). This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, von Willebrand factor (VWF), and fibronectin. Affinity modulation can be monitored in living cells with engineered monovalent Fab fragments derived from ligand-mimetic monoclonal antibodies.35,50,137  Plasma fibrinogen and VWF support platelet aggregation at low and high shear rates, respectively, by bridging αIIbβ3 receptors on adjacent platelets.3  Studies in mice deficient in fibrinogen and VWF indicate that plasma fibronectin can also promote thrombus initiation, growth, and stability at high shear rates.138  (B) Outside-in signaling. Extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tails. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.

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Basic research conducted in the past 3 decades on many facets of αIIbβ3 structure and function has led to remarkable breakthroughs culminating in the development of a chimeric anti-αIIbβ3 monoclonal antibody and small-molecule receptor antagonists now used parenterally to limit the formation of occlusive platelet thrombi in acute cardiovascular indications.5,6  On the other hand, clinical trials of oral αIIbβ3 antagonists have been disappointing and suggest that long-term extracellular blockade of ligand binding to αIIbβ3 might even be dangerous. Conceivably, then, further basic studies of this proven therapeutic target, and of αIIbβ3 signaling in particular, might lead to newer and better ways to diagnose, prevent, or treat arterial thrombosis or other consequences of αIIbβ3 dysfunction. The purpose of this review is to highlight recent experimental and conceptual advances in the integrin field that are particularly relevant to αIIbβ3 and platelets. It will draw from structural analyses of integrins and studies of human and mouse platelets, with the caution that platelets from these 2 species are similar but not identical.7-9  Several excellent general reviews of integrin signaling are also available.4,10-16 

Structure of the αIIbβ3 extracellular domain

Our first glimpse into αIIbβ3 structure was provided nearly 20 years ago when approximately 230-kDa αIIbβ3 complexes were purified from detergent-solubilized platelet membranes and visualized by electron microscopy.17  Rotary-shadowed, negatively stained images revealed an approximately 23-nm (230-Å) complex consisting of an approximately 8-nm (80 Å) globular head and 2 approximately 16-nm (160 Å) flexible stalks. In the absence of detergent, αIIbβ3 aggregated into rosettes that appeared to be contacting each other at the tips of their stalks (Figure 2A-B). Following the cloning of αIIb and β3 and using epitope-mapped antibodies, Weisel and colleagues18  correctly deduced that the stalks contain the C-terminal, transmembrane domain-containing segment of each subunit, and the globular head contains the N-terminal portions. These investigators also found that fibrinogen, von Willebrand factor (VWF), and fibronectin interact with the globular head (Figure 2C-D), thus identifying this domain as containing the ligand contact site. These findings were corroborated by the demonstration of fibrinogen binding to a recombinant αIIbβ3 head domain lacking the β3 stalk.19 

Figure 2.

Rotary-stained electron micrographic images of αIIbβ3. The complex is shown in the absence of detergent (A) or bound to fibrinogen (C). Schematic representations of each are shown in panels B and D. Note that the integrin stalks containing the hydrophobic transmembrane domains have a tendency to self-associate, while the head domain (arrows) binds fibrinogen (red outline). Adapted from Carrell et al17  and Weisel et al18 with permission.

Figure 2.

Rotary-stained electron micrographic images of αIIbβ3. The complex is shown in the absence of detergent (A) or bound to fibrinogen (C). Schematic representations of each are shown in panels B and D. Note that the integrin stalks containing the hydrophobic transmembrane domains have a tendency to self-associate, while the head domain (arrows) binds fibrinogen (red outline). Adapted from Carrell et al17  and Weisel et al18 with permission.

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A number of early models of integrin structure, such as the one shown in the top left panel of Figure 3, were developed based on electron microscopic images, peptide and epitope mapping, photo-affinity and chemical cross-linking, and biochemical analyses of cysteine disulfide-bonding patterns. Portrayed were such structural features as (1) an αIIb subunit composed of an approximately 120-kDa heavy-chain disulfide bonded to an approximately 23-kDa light chain; (2) 4 αIIb calcium-binding domains; (3) a small N-terminal cysteine-rich domain in β3 attached by a disulfide bond to “cysteine-rich repeats” within the body of the molecule; and (4) a large, protease-sensitive “disulfide-bonded loop” within β3 bounded by residues 121 and 348 and containing the major ligand-binding sites.

Figure 3.

Structure of αIIbβ3. An early model of the αIIbβ3 complex (top left) illustrates a number of relevant functional and structural features, including major ligand contact sites (within the yellow rectangle), and the calcium binding region and interchain and intrachain disulfide bonds in αIIb (GPIIb; blue). The β3 subunit is shown in red with its 5 cysteine-rich regions, 1 at the N-terminus and 4 in the stalk, ligand contact sites (yellow rectangle), and 2 chymotrypsin-sensitive cleavage sites (jagged line) that remove the ligand-binding segment, now termed A. Domains visible in the crystal structure of the closely related αVβ3 (top right and bottom panels) are shown in detail and discussed in the text. The PSI domain (bottom left) is depicted schematically because its structure has not been determined. Adapted from Xiong et al20  and Newman139 with permission.

Figure 3.

Structure of αIIbβ3. An early model of the αIIbβ3 complex (top left) illustrates a number of relevant functional and structural features, including major ligand contact sites (within the yellow rectangle), and the calcium binding region and interchain and intrachain disulfide bonds in αIIb (GPIIb; blue). The β3 subunit is shown in red with its 5 cysteine-rich regions, 1 at the N-terminus and 4 in the stalk, ligand contact sites (yellow rectangle), and 2 chymotrypsin-sensitive cleavage sites (jagged line) that remove the ligand-binding segment, now termed A. Domains visible in the crystal structure of the closely related αVβ3 (top right and bottom panels) are shown in detail and discussed in the text. The PSI domain (bottom left) is depicted schematically because its structure has not been determined. Adapted from Xiong et al20  and Newman139 with permission.

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Recent determination of the crystal structure of the extracellular segment of αVβ3 has provided a major advance.20  Remarkably, many of the structural and functional domains predicted in earlier models are recognizable in the 12 domains identified in the crystal, albeit with much higher resolution, and with some notable surprises. As shown in Figure 3, the 4 Ca2+-binding domains in αV are part of a β-propeller, the structure of which had been predicted.21  A large immunoglobulin-like “thigh” domain comprises the remainder of the αV subunit's contribution to the integrin headpiece. In β3, the N-terminal cysteine-rich segment has become the plexin/ semaphorin/integrin (PSI) domain, while the former ligand-binding large disulfide-bonded loop emerges from a discontinuous, immunoglobulin-like hybrid domain in the form of an adhesive “I-like” or A domain. Finally, the previously observed αV stalk is now composed of 2 rigid “calf” modules, and the former cysteine-rich repeats of the β3 stalk have morphed into 4 endothelial growth factor (EGF)-like domains, which, together with a novel flowerlike structure termed the β-terminal domain (βTD), completes the stalk. Comparison of the predicted structures of αIIbβ3 with those actually found in the αVβ3 crystal can be found in Table 1.

Several groups have attempted to reconcile the αVβ3 crystal structure with electron microscopic images analyzed with refined methods.15,22  In one example, electron cryomicroscopy was used to derive a 2-nm (20-Å) resolution Fourier shell transformation density map of hydrated αIIbβ3 complexes frozen in a low-affinity, unliganded state. Taking liberties to introduce a few kinks into the flexible hinge regions within the 2 subunits, the authors were able to visually fit the 12 domains of αIIbβ3 into the extracellular region of their 3-dimensional contour map. They suggest that their model may represent the structure of αIIbβ3 as it exists in the surface membrane of resting platelets.22 

Structure of αIIbβ3 transmembrane and cytoplasmic domains

Since the short cytoplasmic tails of αIIb and β3 play key roles in signaling, much attention has focused on whether they have an ordered 3-dimensional structure and how they might interact with each other and with intracellular proteins. Vinogradova et al23  were the first to determine a nuclear magnetic resonance (NMR) structure for a membrane-anchored form of an isolated αIIb tail and found that it formed an N-terminal α-helix followed by a turn predicted to straighten out upon integrin activation. Ulmer et al24  employed NMR spectroscopy to analyze the αIIb and β3 cytoplasmic tails in aqueous solution and found them to be largely unstructured, with a tendency for the N-terminus of β3 to form a helix and the downstream NPLY747 motif to form a reverse turn that could support talin binding. Interestingly, neither these authors nor Li and colleagues25  could find any evidence for interaction between the αIIb and β3 tails themselves, despite the fact that the latter group expressed the tails in a relatively native state—that is, attached to their respective transmembrane domains buried in phospholipid. Rather, the transmembrane domains, which were present in a largely α-helical conformation, actually promoted the formation of homodimers and homotrimers. Based on the observation that certain mutations within the β3 transmembrane domain enhance the tendency to form αIIb-αIIb and β3-β3-β3 homomeric associations, while at the same time conferring constitutive fibrinogen-binding activity, these workers proposed that homomeric transmembrane helix associations might drive subunit reshuffling to increase receptor clustering and avidity for ligand.26  On the other hand, cysteine scanning mutagenesis of the αIIb and β3 transmembrane domains in the context of the full-length integrin expressed in model cell systems suggests that interactions through a specific heterodimer interface help to maintain the default low-affinity state of the integrin. Furthermore, separation of the transmembrane helices is linked to an increase in αIIbβ3 affinity for ligands.27  Since a portion of the integrin residues involved in the transmission of bidirectional signals are embedded in the plasma membrane,28,29  the structure and function of the αIIbβ3 transmembrane domains clearly warrant further investigation.

In contrast to the above studies, several others have found evidence that integrin cytoplasmic tails can and do interact with each other, and in some cases the nature of the interaction appears to change as a consequence of inside-out activation. A tightly packed approximately 3-nm (30-Å) long cylindrical rod was observed in the region of the αIIbβ3 transmembrane domain by electron cryomicroscopy, corresponding to a pair of tightly packed, parallel, right-handed α-helical coils.22  The cytoplasmic domains could be seen extending from the transmembrane α-helix as a single, cohesive, heart-shaped density of approximately 7 kDa. Two recent NMR structures also show extensive, although somewhat contradictory, interactions between αIIb and β3 cytoplasmic tails near the membrane-proximal interface of each tail.30,31  The latter structure showed intersubunit contacts composed of hydrophobic and hydrostatic interactions mediated by amino acid residues highly conserved among integrins. A recent study has shown that in the presence of membrane-mimetic micelles, the cytoplasmic faces of the αIIb and β3 cytoplasmic tails and the NPLY region of β3 become embedded in the membrane, a conformation that differs importantly from that observed in a strictly aqueous environment. Furthermore, the binding of purified talin to β3 caused un-clasping of the tails and changes in tail-membrane interactions (Figure 4A).32  These in vitro results are largely consistent with fluorescence resonance energy transfer (FRET) studies of green fluorescence protein (GFP)-tagged αL and yellow fluorescent protein (YFP)-tagged β2 subunits in living cells.33  Based on changes in FRET, the cytoplasmic tails were calculated to be close to each other in resting cells but become separated by up to 10 nm (100 Å) following either ligand binding to αLβ2 or agonist-induced cellular activation. Overall, these data provide compelling evidence that intersubunit cytoplasmic tail associations, and possibly heterodimeric transmembrane associations, function to maintain the αIIbβ3 complex in a resting, nonadhesive conformation, while disruption of these interactions causes separation of the tails and propagated changes in the extracellular domains to increase αIIbβ3 affinity.

Figure 4.

Integrin tail-membrane interactions and high-affinity ligand binding. (A) NMR-derived model of αIIb (blue) and β3 (red) cytoplasmic tails. In resting cells (left), the 2 tails contact each other and are also embedded in the membrane via their N-terminal α-helices and the “middle” NPLY region of β3. Under these conditions, talin is not bound to β3. When cells and talin are activated (right), the head domain of talin (H) is released from inhibition by its rod domain (R) and binds to β3. This disrupts the relatively weak integrin tail-tail and tail-membrane interactions, leading to splaying of the tails and bidirectional signaling. Changes similar to those induced by talin binding may be induced by the binding of fibrinogen to αIIbβ3. From Vinogradova et al32  with permission. (B) The deadbolt model of inside-out integrin activation. In the nonactivated integrin, the elongated CD loop of βTD is in close proximity to the βA domain, allowing it to effectively “deadbolt” the F/α7 loop in place, preventing ligands (transparent blue circle) from making contact with βA residues necessary for high-affinity binding. Inside-out signaling is hypothesized to induce conformational changes in the cytoplasmic tails that when transmitted through the transmembrane domains would unlock the deadbolt. The resulting loss of constraints imposed by the CD loop would allow the F/α7 loop to rock back (exaggerated as shown) from the ligand contact site, making the latter available for binding. Certain LIBS antibodies may also move the deadbolt, promoting ligand binding independent of inside-out signals. Adapted from Xiong et al14  with permission.

Figure 4.

Integrin tail-membrane interactions and high-affinity ligand binding. (A) NMR-derived model of αIIb (blue) and β3 (red) cytoplasmic tails. In resting cells (left), the 2 tails contact each other and are also embedded in the membrane via their N-terminal α-helices and the “middle” NPLY region of β3. Under these conditions, talin is not bound to β3. When cells and talin are activated (right), the head domain of talin (H) is released from inhibition by its rod domain (R) and binds to β3. This disrupts the relatively weak integrin tail-tail and tail-membrane interactions, leading to splaying of the tails and bidirectional signaling. Changes similar to those induced by talin binding may be induced by the binding of fibrinogen to αIIbβ3. From Vinogradova et al32  with permission. (B) The deadbolt model of inside-out integrin activation. In the nonactivated integrin, the elongated CD loop of βTD is in close proximity to the βA domain, allowing it to effectively “deadbolt” the F/α7 loop in place, preventing ligands (transparent blue circle) from making contact with βA residues necessary for high-affinity binding. Inside-out signaling is hypothesized to induce conformational changes in the cytoplasmic tails that when transmitted through the transmembrane domains would unlock the deadbolt. The resulting loss of constraints imposed by the CD loop would allow the F/α7 loop to rock back (exaggerated as shown) from the ligand contact site, making the latter available for binding. Certain LIBS antibodies may also move the deadbolt, promoting ligand binding independent of inside-out signals. Adapted from Xiong et al14  with permission.

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Conformational changes associated with β3 integrin activation

Several observations indicate that the extracellular domains of αIIbβ3 and αVβ3 must undergo conformational transitions upon cellular activation or ligand binding. First, activation of platelets or endothelial cells induces the binding of arginine-glycine-aspartic acid (RGD)-containing antibody Fab fragments to αIIbβ3 and αVβ3, respectively.34,35  In the case of αIIbβ3, Fab binding requires discontinuous regions of the αIIb β-propeller and the β3 A domain.36  Second, when platelets are activated by agonists, a change in FRET is observed between fluorophore-conjugated antibodies bound to αIIb and β3.37  Finally, anti-αIIb or anti-β3 antibodies of the ligand-induced binding sites (LIBS) type preferentially recognize the ligand-occupied form of the integrin, and they in turn increase receptor affinity. In terms of primary amino acid sequence, the epitopes for many of these LIBS antibodies are located a relatively long distance from the ligand-binding headpiece, suggesting that the integrin is subject to long-range conformational changes.38 

Differences in αVβ3 structure have been visualized in negatively stained electron microscopic images of the integrin in apparent low- and high-affinity states.39  The low-affinity, unliganded form was present as a compact, V-shaped structure highly reminiscent of the bent conformation found in the crystal structure of αVβ3 (Figure 3), becoming extended upon ligand binding into the “head + 2 tails” configuration previously visualized by other investigators (eg, Figure 2). This transition has been described as analogous to a “switchbladelike” movement such that the extended form may represent the ligand-bound high-affinity receptor. However, a less drastic change may be all that is required for initial transformation of β3 integrins from low- to high-affinity state. Coined the “deadbolt” model,14  inside-out signals are postulated to transmit conformational changes through the transmembrane helices into the immediately proximal βTD. The CD loop of the βTD, which in resting integrins acts like a deadbolt to pin the F/α7 loop of the βA domain forward to obstruct contact with macromolecular ligands (Figure 4B), then moves out of the way, allowing the F/α7 loop to swing away from the ligand contact site, making it available for productive, high-affinity ligand binding.

Experimental evidence that displacement of the F/α7 loop is involved in ligand binding comes from studies of αLβ2, where mutational shortening of the β2 α-helix by approximately one turn resulted in a constitutively active receptor.40  One of the most attractive features of this model is that larger-scale structural changes are not required to convert β3 integrins into high-affinity receptors. Furthermore, the model does not preclude switchbladelike straightening of the bent integrin or separation of the cytoplasmic tails, both of which are likely to promote outside-in signaling in response to ligand binding. Additional studies are required to rigorously test and refine existing models of integrin activation and, in particular, to fully understand coordinated transitions among the integrin extracellular, transmembrane, and cytoplasmic domains, molecular movements that will likely affect outside-in as well as inside-out signaling.

Integrin clustering

In addition to conformational changes, cell activation promotes the lateral mobility and clustering of integrins within the plane of the plasma membrane.41  Initially, small oligomers or “microclusters” below the resolution of the light microscope may contribute to “avidity” or “valency” regulation of ligand binding.15,42  Microclustering in native membranes or living cells has been difficult to study, but new techniques are beginning to open up this area of investigation.33,43  The αIIbβ3 clustering may be promoted by several mechanisms, including the binding of multivalent ligands,43,44  ligand self-association,45,46  lateral interactions of integrins with other membrane proteins,47  reversible integrin linkages to the actin cytoskeleton,48  and homomeric interactions of the transmembrane domains.26  Integrin conformational change and clustering are not mutually exclusive; they are complementary and may even be mechanistically linked. Each may be involved in different aspects of bidirectional signaling. For example, conformational change seems to be the dominant way in which ligand binding to αIIbβ3 and αVβ3 is regulated, while clustering is important in triggering activation of Src and Syk protein tyrosine kinases during outside-in signaling.49-51 

Regulation of inside-out signaling: excitatory and inhibitory agonist receptors

Binding of adhesive ligands to αIIbβ3 can be triggered by soluble agonists, such as adenosine diphosphate (ADP), thrombin, epinephrine, and thromboxane A2, which engage cognate G-protein-coupled receptors.9  In addition, certain platelet adhesion receptors, notably GPIb-IX-V (the primary receptor for VWF), GPVI (collagen), α2β1 (collagen), and even αIIbβ3, can trigger activation signals when bound to and clustered by ECM ligands.3,52-54  The relative contribution of soluble and ECM stimuli to inside-out signaling likely varies with flow conditions and other circumstances of vascular injury. For example, GPIb-IX-V function is most relevant under conditions of high shear typical of the arteriolar and capillary circulations and in stenotic arteries.3  An important but under-studied process is inhibition or reversal of ligand binding to αIIbβ3. Activation of αIIbβ3 is negatively regulated in a complex manner by cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP), generated by interaction of platelets with prostacyclin (PGI2) and nitric oxide (NO), respectively,9  and by an endothelial cell ecto-ADPase (CD39).55  In mouse platelets, the excitatory function of GPVI and GPIb-IX-V is partly dependent on the associated FcR γ-chain, whose tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) help recruit tyrosine kinases to these receptors.53,54  Signaling through GPVI and GPIb-IX-V is dampened by platelet endothelial cell adhesion molecule 1 (PECAM-1), an immunoglobulin superfamily receptor whose immunoreceptor tyrosine-based inhibitory motifs (ITIMs) recruit SHP-1 (Src homology 2 [SH2] domain-containing tyrosine phosphatase 1) and SHP-2 tyrosine phosphatases.56-58 

Signaling intermediates that link agonist receptors to αIIbβ3

Receptors couple to second messengers such as Ca2+, cyclic nucleotides, and products of phospholipases and tyrosine kinases.9,53  A major gap remains in how second messengers effect functional changes in αIIbβ3. For example, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI 3-kinase), and Rap1b have been implicated as intermediates in promoting inside-out signaling, but the identities and activities of the relevant effectors of these enzymes remain to be determined.9,59  Rap1b serves as a timely case in point.

Rap1 is a member of the Ras family of small guanosine triphosphatases (GTPases) and has been implicated generally in promoting cell adhesion and migration through effects on affinity and/or avidity modulation of integrins.60  Rap1b, the predominant isoform in platelets, cycles from a guanosine diphosphate (GDP)-bound inactive state to a GTP-bound active state upon addition of agonists to Gi-coupled receptors,61,62  binding of collagen to GPVI,63  and even binding of fibrinogen to αIIbβ3.64  Rap1b associates with the actin cytoskeleton of activated platelets and is a substrate for protein kinase A, although the effect of this phosphorylation is unknown.64  A link between Rap1b and affinity modulation of αIIbβ3 has been established in primary murine megakaryocytes.35,65  Overexpression of a constitutively active Rap1b mutant or of CalDAG-GEFI, a Rap exchange factor, potentiates agonist-induced fibrinogen binding to αIIbβ3, and this effect is blocked by inhibitors of actin polymerization. Overexpression of Rap1-GTPase-activating protein (Rap1-GAP), which converts Rap1-GTP to Rap1-GDP, partially blocks agonist-induced fibrinogen binding, suggesting that endogenous Rap1b promotes, but is not sufficient for, inside-out signaling, perhaps through some effect on the actin cytoskeleton. This interpretation is consistent with the recent observation that Rap1b-deficient mouse platelets undergo reduced aggregation responses to agonists.66 

Interestingly, CalDAG-GEFI contains a C1 domain that may bind diacylglycerol and an EF hand domain that binds Ca2+.60  Consequently, generation of these second messengers by phospholipase C could provide one means by which Rap1b activity is regulated in platelets. However, other exchange factors for Rap1b have been identified60  and some may be expressed in platelets. Rap1b effectors involved in αIIbβ3 signaling have yet to be identified. In this context, RAPL is a Rap1-GTP-binding protein that reportedly coimmunoprecipitates with and clusters αLβ2 when T lymphocytes are activated by chemokines.67  Its presence and function in platelets have not been determined.

Proximal regulation of αIIbβ3 by integrin-binding proteins

In theory, ligand binding to αIIbβ3 could be regulated by integrin interactions with intracellular, extracellular, or transmembrane molecules. RGD-containing ligands, even short peptides, can stabilize the high-affinity conformation of purified αIIbβ3.68  In platelets, MnCl2 or LIBS antibodies convert αIIbβ3 into a high-affinity conformation.69  Some have speculated that certain αIIbβ3 antagonists or soluble CD40 ligand may exert similar effects in vivo6,70  and that some drug-dependent anti-αIIbβ3 antibodies may recognize LIBS epitopes exposed by binding of the drug.71,72  Reducing agents such as dithioethreitol can activate purified or platelet αIIbβ3, as can mutation of single cysteines within the β3 EGF repeats.73  The platelet surface and β3 integrins in particular are reported to possess thiol isomerase activity,74,75  leading to the proposition that disulfide exchange may help to regulate αIIbβ3 activation.76,77  Also, a pool of αIIbβ3 may form complexes with CD9 (a tetraspanin)47  or CD47 (a thrombospondin receptor).78  The relationship between CD47 and αIIbβ3 appears particularly complex. Specific peptides from thrombospondin can bind to CD47 and activate platelet G proteins, providing one way for CD47 to regulate αIIbβ3.78  In addition, platelet CD47 can interact with receptors in activated endothelial cells, leading to αIIbβ3 activation.79  Finally, the extracellular portion of CD47 bound to a thrombospondin peptide can directly modulate αIIbβ3 activation state in Chinese hamster ovary (CHO) cells.80  Despite these observations, no platelet aggregation abnormalities have been reported in CD47-deficient mice.

Evidence to date indicates that any role for extracellular or transmembrane molecules in affinity modulation is secondary to αIIbβ3 regulation by intracellular proteins, and in particular talin, which engage the integrin cytoplasmic tails. Talin1 is an approximately 270-kDa antiparallel dimer composed of an approximately 50-kDa N-terminal FERM domain, which contains F1-3 subdomains and assumes a phosphotyrosine-binding (PTB) domain-like fold, and an approximately 220-kDa C-terminal rod domain (Figure 5).81-83  F2-3 contains a major binding site for integrin β cytoplasmic tails and several other proteins, including type Iγ phosphatidylinositol phosphate kinase (PIPKIγ), an enzyme responsible for generating the lipid second messenger, PIP2.84  The rod domain, separated from the FERM domain by a calpain cleavage site, contains the major binding sites for F-actin.

Figure 5.

Domain structure of talin, a key protein in regulation of inside-out integrin signaling. Location of binding sites for other proteins is depicted.

Figure 5.

Domain structure of talin, a key protein in regulation of inside-out integrin signaling. Location of binding sites for other proteins is depicted.

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Interest in talin as an integrin regulator comes from several lines of investigation. First, talin (or F2-3) binds specifically to integrin β tails in vitro, including β1, β2, and β3.82,85-88  Second, overexpression of the FERM or F2-3 domains activates αIIbβ3 in CHO cells.82,86,88  Third, as depicted in Figure 4A, NMR, x-ray crystallographic, and FRET analyses are consistent with the idea that talin interacts with the N-terminus and midportion of β tails, thereby splaying the β and α tails and modifying their interactions with the membrane.31,33,89,90  Fourth, mutations in talin F2-3 or β tails predicted to disrupt their interaction also eliminate integrin activation in CHO cells.88  Thus, the inability to bind talin may explain the thrombasthenic phenotype of human platelets where the membrane-distal half of the β3 tail has been deleted.91  Finally, knockdown of talin in CHO cells and megakaryocytes by RNA interference ablates energy-dependent activation of β1 and β3 integrins.88 

Important questions remain about the proximal regulation of αIIbβ3. How is talin's recruitment to the platelet membrane and to αIIbβ3 regulated by agonists?92  Perhaps talin transforms from a compact “autoinhibited” conformation to an open conformation in response to activation signals, analogous to several other proteins that influence the actin cytoskeleton, such as vinculin, Wiskott-Aldrich syndrome protein (WASP), and PAK (p21-activated kinase). Signaling events hypothesized to regulate talin include PIP2 binding,93  serine-threonine phosphorylation,94  and proteolysis by calpain.95  In addition, Src-dependent tyrosine phosphorylation of PIPKIγ may increase its ability to compete with integrin β tails for talin,96  and tyrosine phosphorylation of β tails may reduce their binding to talin.94  Does talin regulate integrin avidity as well as affinity? Talin's role in linking integrins to actin filaments, in clustering of integrins into adhesion complexes, and in force generation at the cell-ECM interface certainly place it in a position to do so.81,97,98  Finally, do other integrin cytoplasmic tail-binding proteins regulate αIIbβ3 affinity? Both calcium and integrin binding protein (CIB), which binds to αIIb, and β3 endonexin, which binds to β3, activate αIIbβ3 in model cell systems.99-101  However, β3 endonexin does not activate αIIbβ3 in the absence of talin,88  and determining the inside-out functions of this and other tail-binding proteins in platelets requires further study.

Regulation of outside-in signaling

Maximal secretory, procoagulant, and clot retraction responses of platelets generally require ligand binding to αIIbβ3 and close platelet-platelet contact. The term “contact-dependent signaling” has been used to describe this phenomenon, in which other ligand-receptor pairs have also been implicated, including ephrin/Eph receptor kinases, CD40 ligand/CD40, and Gas6/Axl-Sky-Mer receptor kinases.70,102-104  The best understood example of contact-dependent signaling is outside-in signaling through αIIbβ3.105 

Platelet adhesion to fibrinogen or VWF triggers morphologic changes ranging from filopodial and lamellipodial extension to full spreading.106-109  As in nucleated cells, these changes are mediated by effectors of the Rho GTPases, cdc42, Rac1, and Rho A.107,110  The morphologic changes are associated with dynamic modifications of the actin cytoskeleton that affect the polymerization state and organization of actin.109,111  αIIbβ3 participates in this process by nucleating signaling complexes at adhesion sites that regulate actin.106,109,112,113  Platelets and αIIbβ3-expressing CHO cells adherent to fibrinogen have frequently been used as model systems to study this process. In both cases, outside-in signaling occurs in a discrete pattern whereby ligand binding initiates integrin clustering and assembly of a nascent signaling complex proximal to the cytoplasmic tails of αIIbβ3, followed by the growth of a larger actin-based signaling complex.

Initiation of outside-in signaling

Among the earliest detectable biochemical responses of platelets to fibrinogen binding is activation of Src and Syk protein tyrosine kinases.112  Occupancy of αIIbβ3 by fibrinogen causes integrin microclustering,43,44  which appears necessary for this tyrosine kinase activation.49,51  Although some monovalent ligands promote αIIbβ3 homo-oligomerization in detergent solution,114  there is no unequivocal evidence yet that they do so or trigger outside-in signaling in vivo. Soluble CD40 ligand can bind and induce outside-in signaling through αIIbβ3, but it is a trimer, suggesting even in this case that clustering of αIIbβ3 is required.70,115 

Components of a nascent αIIbβ3 signaling complex have been identified in platelets and CHO cells based on their ability to coimmunoprecipitate with αIIbβ3 or to become rapidly tyrosine phosphorylated by integrin-associated Src or Syk, even in the presence of inhibitors of actin polymerization.112  Buttressed by studies with purified proteins as well as by detection of specific protein-protein interactions in living cells,116  the following sequence of events for assembly of the αIIbβ3 signaling complex can be envisioned. (1) Src kinases constitutively bound to the β3 cytoplasmic tail become activated when fibrinogen engages and clusters αIIbβ351,112  (Figure 6). (2) Syk is recruited to the β3 tail and activated by Src.112,117  (3) Src and/or Syk phosphorylate substrates, including SLP-76, ADAP and c-Cbl (molecular adaptors), and Vav (a Rac GTPase), that are implicated in signaling to the actin cytoskeleton.118-120 

Figure 6.

Model for αIIbβ3 regulation of Src. (Left) According to current structural models,140  Src family kinases are membrane associated and maintained in a “clamped,” inactive state through intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine motif at Tyr529 (Y529), and the SH3 domain and a polyproline motif in the linker region between the SH2 domain and the N-lobe of the catalytic domain. (Middle) In platelets, a pool of c-Src (and several other Src family kinases) is constitutively bound to αIIbβ3 through interaction of the β3 cytoplasmic tail with the SH3 domain. This may maintain Src in a partially unclamped, primed state but not yet fully active, in part because Tyr529 remains phosphorylated by integrin-associated Csk. (Right) Upon αIIbβ3 ligation, Src becomes clustered and Csk dissociates from the integrin complex. The net result is dephosphorylation of Tyr529 by an unidentified tyrosine phosphatase and autophosphorylation of Tyr418 (Y418) in the Src activation loop. Consequently, Src is now unclamped and fully active to phosphorylate downstream effectors. From Arias-Salgado et al51  and Obergfell et al112  with permission.

Figure 6.

Model for αIIbβ3 regulation of Src. (Left) According to current structural models,140  Src family kinases are membrane associated and maintained in a “clamped,” inactive state through intramolecular interactions between the SH2 domain and a C-terminal phosphotyrosine motif at Tyr529 (Y529), and the SH3 domain and a polyproline motif in the linker region between the SH2 domain and the N-lobe of the catalytic domain. (Middle) In platelets, a pool of c-Src (and several other Src family kinases) is constitutively bound to αIIbβ3 through interaction of the β3 cytoplasmic tail with the SH3 domain. This may maintain Src in a partially unclamped, primed state but not yet fully active, in part because Tyr529 remains phosphorylated by integrin-associated Csk. (Right) Upon αIIbβ3 ligation, Src becomes clustered and Csk dissociates from the integrin complex. The net result is dephosphorylation of Tyr529 by an unidentified tyrosine phosphatase and autophosphorylation of Tyr418 (Y418) in the Src activation loop. Consequently, Src is now unclamped and fully active to phosphorylate downstream effectors. From Arias-Salgado et al51  and Obergfell et al112  with permission.

Close modal

Propagation of outside-in signaling

As the nascent complex assembles, many additional proteins are recruited that are capable of influencing actin dynamics and reorganization. These include Rac, Nck (an adapter), PAK, PI 3-kinase, and vasodilator-stimulated phosphoprotein (VASP), an actin-bundling protein (Figure 7). Although not all components of the signaling network have been identified, 3 proteins warrant particular discussion here because each is tyrosine phosphorylated by Src and/or Syk during platelet aggregation and spreading and each probably helps to morph nascent complexes into actin-based complexes. These proteins are β3 itself, phospholipase Cγ, and α-actinin. Phosphorylation of the β3 cytoplasmic tail at residues 747 and 759 may enhance post-ligand-binding events by generating docking sites for SH2-containing protein (Shc), an adapter in Ras signaling, and myosin, a motor protein involved in clot retraction and stabilization of platelet aggregates.121,122  Mice in which these tyrosines have been mutated to phenylalanine exhibit rebleeding from tail wounds and subtle defects in clot retraction and platelet aggregation.105,123 

Figure 7.

Cartoon depicting portions of the signaling network linking αIIbβ3 to actin polymerization and reorganization. The insert provides a key to some of the modules or domains within the proteins that mediate or regulate protein functions and/or interactions. Domain abbreviations: CH, calponin homology; P-Tyr, phosphotyrosine; PTB, phosphotyrosine binding; PH, pleckstrin homology; WH, WASP homology; and VH, verprolin homology. WIP indicates WASP-interacting protein; PLCγ, phospholipase Cγ. The figure is offered solely to provide a visual context for the discussion in the text of early phases of outside-in signaling. No attempt is made to show all proteins involved or all interactions of a given protein, and important signaling cross-talk between αIIbβ3 and other platelet receptors is not depicted.9  See Bearer et al,109  Hartwig et al,111  and Calderwood et al124  for reviews of integrin-dependent actin dynamics and organization in platelets.

Figure 7.

Cartoon depicting portions of the signaling network linking αIIbβ3 to actin polymerization and reorganization. The insert provides a key to some of the modules or domains within the proteins that mediate or regulate protein functions and/or interactions. Domain abbreviations: CH, calponin homology; P-Tyr, phosphotyrosine; PTB, phosphotyrosine binding; PH, pleckstrin homology; WH, WASP homology; and VH, verprolin homology. WIP indicates WASP-interacting protein; PLCγ, phospholipase Cγ. The figure is offered solely to provide a visual context for the discussion in the text of early phases of outside-in signaling. No attempt is made to show all proteins involved or all interactions of a given protein, and important signaling cross-talk between αIIbβ3 and other platelet receptors is not depicted.9  See Bearer et al,109  Hartwig et al,111  and Calderwood et al124  for reviews of integrin-dependent actin dynamics and organization in platelets.

Close modal

Phospholipase Cγ is a substrate of Src and Bruton tyrosine kinase (Btk) kinases, and its activation by tyrosine phosphorylation downstream of αIIbβ3 generates some of the diacylglycerol and inositol phosphate (IP3) needed for maximal platelet aggregation and spreading.113  Diacylglycerol and IP3-dependent Ca2+ fluxes activate conventional and novel isoforms of PKC discussed previously in the context of inside-out signaling. Preliminary studies show that certain PKC isoforms coimmunoprecipitate with αIIbβ3 under some conditions, and broad-spectrum PKC inhibitors block platelet spreading on fibrinogen (Churito Buensuceso, Alessandra Soriani, Achim Obergfell, Koji Eto, and S.J.S., unpublished observations, January 2004). Thus, some integrin-associated proteins may regulate both phases of αIIbβ3 signaling.

α-actinin is a homodimeric actin-binding protein that localizes to integrin adhesion sites. The nonmuscle isoform found in platelets contains binding sites for vinculin, zyxin, and the membrane-proximal portions of β1, β2, and β3 integrin cytoplasmic tails.124  Overexpression of full-length α-actinin in fibroblasts leads to stabilization of adhesion sites, while integrin-binding fragments disrupt actin stress fibers, focal adhesions, and mechanotransduction.125  α-actinin becomes tyrosine phosphorylated on a single N-terminal residue in response to platelet aggregation or spreading.126  Phosphorylation may be mediated by focal adhesion kinase (FAK), whose own activation is dependent on actin polymerization following platelet costimulation through agonist and αIIbβ3 receptors. In a model system, tyrosine phosphorylation of α-actinin reduced its cosedimentation with F-actin.126  Therefore, phosphorylation of α-actinin during later stages of outside-in signaling might regulate αIIbβ3 linkages with actin and the assembly/disassembly of actin-based signaling complexes.

Many facets of outside-in αIIbβ3 signaling remain to be explored. What are the identities and functions of the phosphatases that counterbalance the effects of the protein and lipid kinases that operate in integrin signaling? What are the roles in platelets of other proteins, such as skelemin and integrin-linked kinase, reported to interact with the αIIb or β3 cytoplasmic tails in model systems?101,127,128  Disassembly of αIIbβ3-based signaling complexes may be required to achieve full platelet spreading or to limit platelet adhesion and aggregation to the hemostatic plug. How is disassembly regulated? Perhaps it involves αIIbβ3-dependent activation of FAK and its effectors.129,130  Perhaps it involves specific protein or lipid phosphatases or proteases like calpain, which cleave β3, Src, and other proteins upon platelet aggregation. Do the other integrins in platelets engage in bidirectional signaling? Recent investigations of αVβ3 and α2β1 suggest that they do.53,131-133  How are they regulated?

αIIbβ3 was identified as the platelet fibrinogen receptor over 25 years ago. Ensuing investigations of αIIbβ3 and its relative, αVβ3, have provided many key insights about integrin structure and function. Some of these have led to improvements in clinical practice, most notably the use of parental αIIbβ3 antagonists to prevent arterial thrombosis. Future studies of αIIbβ3 signaling promise to yield additional information of clinical relevance. For example, while deficiency of αIIbβ3 is rare, specific defects in αIIbβ3-related signal transduction may account for many incompletely characterized bleeding disorders associated with defects in platelet aggregation.134,135  Moreover, currently available antiplatelet drugs, such as aspirin and clopidogrel, work in effect by dampening inside-out signaling to αIIbβ3.6  Although bidirectional αIIbβ3 signaling is complex, novel orally active drugs may eventually be developed that target specific facets of this process. More generally, progress in integrin research can also be anticipated in several other areas of interest to hematologists, among them elucidation of the relationships between integrin polymorphisms, platelet function, and thrombotic risk; the pathogenesis of immune thrombocytopenias; the role of αIIbβ3 in hematopoietic stem cells136 ; and the development of drugs that modulate integrin signaling in inflammatory diseases and cancer. Stay tuned.

Prepublished online as Blood First Edition Paper, June 17, 2004; DOI 10.1182/blood-2004-04-1257.

Supported by grants from the National Heart Lung and Blood Institute.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

The authors are indebted to the many colleagues in the integrin field, basic scientists and clinicians alike, who have made fundamental contributions to the work and concepts summarized here.

1
Wagner DD, Burger PC. Platelets in inflammation and thrombosis.
Arterioscler Thromb Vasc Biol
.
2003
;
23
:
2131
-2137.
2
Frenette PS, Denis CV, Weiss L, et al. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo.
J Exp Med
.
2000
;
191
:
1413
-1422.
3
Ruggeri ZM. Platelets in atherothrombosis.
Nat Med
.
2002
;
8
:
1227
-1234.
4
Hynes R. Integrins: bidirectional, allosteric signaling machines.
Cell
.
2002
;
110
:
673
-687.
5
Coller BS. Anti-GPIIb/IIIa drugs: current strategies and future directions.
Thromb Haemost
.
2001
;
86
:
427
-443.
6
Bhatt DL, Topol EJ. Scientific and therapeutic advances in antiplatelet therapy.
Nat Rev Drug Discov
.
2003
;
2
:
15
-28.
7
Tsakiris DA, Scudder L, Hodivala-Dilke K, Hynes RO, Coller BS. Hemostasis in the mouse (Mus musculus): a review.
Thromb Haemost
.
1999
;
81
:
177
-188.
8
Sambrano GR, Weiss EJ, Zheng YW, Huang W, Coughlin SR. Role of thrombin signalling in platelets in haemostasis and thrombosis.
Nature
.
2001
;
413
:
74
-78.
9
Brass LF. Molecular basis for platelet activation. In: Hoffman R, Benz E, Shattil S, et al, eds.
Hematology. Basic Principles and Practice
. 4 th ed. New York, NY: Churchill-Livingstone. In press.
10
Juliano RL. Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin superfamily members.
Annu Rev Pharmacol Toxicol
.
2002
;
42
:
283
-323.
11
Schwartz MA, Ginsberg MH. Networks and crosstalk: integrin signalling spreads.
Nat Cell Biol
.
2002
;
4
:
E65
-E68.
12
Shimaoka M, Takagi J, Springer TA. Conformational regulation of integrin structure and function.
Annu Rev Biophys Biomol Struct
.
2002
;
31
:
485
-516.
13
Travis MA, Humphries JD, Humphries MJ. An unraveling tale of how integrin are activated from within.
Trends Pharmacol Sci
.
2003
;
24
:
192
-197.
14
Xiong JP, Stehle T, Goodman SL, Arnaout MA. New insights into the structural basis of integrin activation.
Blood
.
2003
;
102
:
1155
-1159.
15
Carman CV, Springer TA. Integrin avidity regulation: are changes in affinity and conformation underemphasized?
Curr Opin Cell Biol
.
2003
;
15
:
1
-10.
16
DeMali KA, Wennerberg K, Burridge K. Integrin signaling to the actin cytoskeleton.
Curr Opin Cell Biol
.
2003
;
15
:
572
-582.
17
Carrell NA, Fitzgerald LA, Steiner B, Erickson HP, Phillips DR. Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy.
J Biol Chem
.
1985
;
260
:
1743
-1749.
18
Weisel JW, Nagaswami C, Vilaire G, Bennett JS. Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fi-brinogen and other ligands by electron microscopy.
J Biol Chem
.
1992
;
267
:
16637
-16643.
19
Wippler J, Kouns WC, Schlaeger E-J, Kuhn H, Hadvary P, Steiner B. The integrin αIIbβ3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the β3 subunit.
J Biol Chem
.
1994
;
269
:
8754
-8761.
20
Xiong JP, Stehle T, Diefenbach B, et al. Crystal structure of the extracellular segment of integrin αVβ3.
Science
.
2001
;
294
:
339
-345.
21
Springer TA. Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain.
Proc Natl Acad Sci U S A
.
1997
;
94
:
65
-72.
22
Adair BD, Yeager M. Three-dimensional model of the human platelet integrin αIIbβ3 based on electron cryomicroscopy and x-ray crystallography.
Proc Natl Acad Sci U S A
.
2002
;
99
:
14059
-14064.
23
Vinogradova O, Haas T, Plow EF, Qin J. A structural basis for integrin activation by the cytoplasmic tail of the αIIb-subunit.
Proc Natl Acad Sci U S A
.
2000
;
97
:
1450
-1455.
24
Ulmer TS, Yaspan B, Ginsberg MH, Campbell ID. NMR analysis of structure and dynamics of the cytosolic tails of integrin αIIbβ3 in aqueous solution.
Biochemistry
.
2001
;
40
:
7498
-7508.
25
Li R, Babu CR, Lear JD, Wand AJ, Bennett JS, DeGrado WF. Oligomerization of the integrin αIIbβ3: roles of the transmembrane and cytoplasmic domains.
Proc Natl Acad Sci U S A
.
2001
;
98
:
12462
-12467.
26
Li R, Mitra N, Gratkowski H, et al. Activation of integrin αIIbβ3 by modulation of transmembrane helix associations.
Science
.
2003
;
300
:
795
-798.
27
Luo BH, Springer TA, Takagi J. A specific interface between integrin transmembrane helices and affinity for ligand.
PLoS Biol
.
2004
;
2
:
776
-786.
28
Li R, Babu CR, Valentine K, et al. Characterization of the monomeric form of the transmembrane and cytoplasmic domains of the integrin β3 subunit by NMR spectroscopy.
Biochemistry
.
2002
;
41
:
15618
-15624.
29
Stefansson A, Armulik A, Nilsson I, Von Heijne G, Johansson S. Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits.
J Biol Chem
.
2004
;
279
:
21200
-21205.
30
Weljie AM, Hwang PM, Vogel HJ. Solution structures of the cytoplasmic tail complex from platelet integrin alpha IIb- and beta 3-subunits.
Proc Natl Acad Sci U S A
.
2002
;
99
:
5878
-5883.
31
Vinogradova O, Velyvis A, Velyviene A, et al. A structural mechanism of integrin αIIbβ3 “inside-out” activation as regulated by its cytoplasmic face.
Cell
.
2002
;
110
:
587
-597.
32
Vinogradova O, Vaynberg J, Kong X, Haas TA, Plow EF, Qin J. Membrane-mediated structural transitions at the cytoplasmic face during integrin activation.
Proc Natl Acad Sci U S A
.
2004
;
101
:
4094
-4099.
33
Kim M, Carman CV, Springer TA. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.
Science
.
2003
;
301
:
1720
-1725.
34
Byzova TV, Goldman CK, Pampori N, et al. A mechanism for modulation of cellular responses to VEGF: activation of the integrins.
Mol Cell
.
2000
;
6
:
851
-860.
35
Bertoni A, Tadokoro S, Eto K, et al. Relationships between Rap1b, affinity modulation of integrin αIIbβ3, and the actin cytoskeleton.
J Biol Chem
.
2002
;
277
:
25715
-25721.
36
Puzon-McLaughlin W, Kamata T, Takada Y. Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin αIIbβ3.
J Biol Chem
.
2000
;
275
:
7795
-7802.
37
Sims PJ, Ginsberg MH, Plow EF, Shattil SJ. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex.
J Biol Chem
.
1991
;
266
:
7345
-7352.
38
Du X, Gu M, Weisel J, et al. Long range propagation of conformational changes in integrin αIIbβ3.
J Biol Chem
.
1993
;
268
:
23087
-23092.
39
Takagi J, Petre B, Walz T, Springer T. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling.
Cell
.
2002
;
110
:
599
-611.
40
Yang W, Shimaoka M, Chen J, Springer TA. Activation of integrin beta-subunit I-like domains by one-turn C-terminal alpha-helix deletions.
Proc Natl Acad Sci U S A
.
2004
;
101
:
2333
-2338.
41
Kucik DF. Rearrangement of integrins in avidity regulation by leukocytes.
Immunol Res
.
2002
;
26
:
199
-206.
42
Bazzoni G, Hemler ME. Are changes in integrin affinity and conformation overemphasized?
Trends Biochem Sci
.
1998
;
23
:
30
-34.
43
Buensuceso C, De Virgilio M, Shattil SJ. Detection of integrin αIIbβ3 clustering in living cells.
J Biol Chem
.
2003
;
278
:
15217
-15224.
44
Loftus JC, Albrecht RM. Redistribution of the fi-brinogen receptor of human platelets after surface activation.
J Cell Biol
.
1984
;
99
:
822
-829.
45
Simmons SR, Albrecht RM. Self-association of bound fibrinogen on platelet surfaces.
J Lab Clin Med
.
1996
;
128
:
39
-50.
46
Savage B, Sixma JJ, Ruggeri ZM. Functional self-association of von Willebrand factor during platelet adhesion under flow.
Proc Natl Acad Sci U S A
.
2002
;
99
:
425
-430.
47
Hemler ME. Integrin associated proteins.
Curr Opin Cell Biol
.
1998
;
10
:
578
-585.
48
Bennett JS, Zigmond S, Vilaire G, Cunningham M, Bednar B. The platelet cytoskeleton regulates the affinity of the integrin αIIbβ3 for fibrinogen.
J Biol Chem
.
1999
;
274
:
25301
-25307.
49
Hato T, Pampori N, Shattil SJ. Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin αIIbβ3.
J Cell Biol
.
1998
;
141
:
1685
-1695.
50
Pampori N, Hato T, Stupack DG, et al. Mechanisms and consequences of affinity modulation of integrin αVβ3 detected with a novel patch-engineered monovalent ligand.
J Biol Chem
.
1999
;
274
:
21609
-21616.
51
Arias-Salgado EG, Lizano S, Sarker S, Brugge JS, Ginsberg MH, Shattil SJ. Src kinase activation by a novel and direct interaction with the integrin β cytoplasmic domain.
Proc Natl Acad Sci U S A
.
2003
;
100
:
13298
-13302.
52
Nesbitt WS, Kulkarni S, Giuliano S, et al. Distinct glycoprotein Ib/V/IX and integrin αIIbβ3-dependent calcium signals cooperatively regulate platelet adhesion under flow.
J Biol Chem
.
2002
;
277
:
2965
-2972.
53
Nieswandt B, Watson SP. Platelet collagen interaction: is GPVI the central receptor?
Blood
.
2003
;
102
:
449
-461.
54
Kasirer-Friede A, Cozzi MR, Mazzucato M, De Marco L, Ruggeri ZM, Shattil S. Signaling through GP Ib-IX-V activates αIIbβ3 independently of other receptors.
Blood
.
2004
;
103
:
3403
-3411.
55
Marcus AJ, Broekman MJ, Drosopoulos JHF, et al. The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.
J Clin Invest
.
1997
;
99
:
1351
-1360.
56
Patil S, Newman DK, Newman PJ. Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen.
Blood
.
2001
;
97
:
1727
-1732.
57
Rathore V, Stapleton MA, Hillery CA, et al. PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets.
Blood
.
2003
;
102
:
3658
-3664.
58
Newman PJ, Newman DK. Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology.
Arterioscler Thromb Vasc Biol
.
2003
;
23
:
953
-964.
59
Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation.
J Thromb Haemost
.
2003
;
1
:
1602
-1612.
60
Bos JL, de Rooij J, Reedquist KA. Rap1 signalling: adhering to new models.
Nat Rev Mol Cell Biol
.
2001
;
2
:
369
-377.
61
Woulfe D, Jiang H, Mortensen R, Yang J, Brass LF. Activation of Rap1B by Gi family members in platelets.
J Biol Chem
.
2002
;
277
:
23382
-23390.
62
Lova P, Paganini S, Sinigaglia F, Balduini C, Torti M. A Gi-dependent pathway is required for activation of the small GTPase Rap1b in human platelets.
J Biol Chem
.
2002
;
277
:
131
-138.
63
Larson MK, Chen H, Kahn ML, et al. Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets.
Blood
.
2003
;
101
:
1409
-1415.
64
Franke B, Van Triest M, De Bruijn KMT, et al. Sequential regulation of the small GTPase Rap1 in human platelets.
Mol Cell Biol
.
2000
;
20
:
779
-785.
65
Eto K, Murphy R, Kerrigan SW, et al. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling.
Proc Natl Acad Sci U S A
.
2002
;
99
:
12819
-12824.
66
Wodnicka MM, Fisher TH, Cullen J, et al. Bleeding phenotype and decreased viability in rap1b knockout mice.
J Thromb Haemost
.
2003
;
1
(suppl):
OC213
.
67
Katagiri K, Maeda A, Shimonaka M, Kinashi T. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1.
Nat Immunol
.
2003
;
4
:
741
-748.
68
Du X, Plow EF, Frelinger AL III, O'Toole TE, Loftus JC, Ginsberg MH. Ligands “activate” integrin αIIbβ3 (platelet GPIIb-IIIa).
Cell
.
1991
;
65
:
409
-416.
69
Frelinger AL III, Du X, Plow EF, Ginsberg MH. Monoclonal antibodies to ligand-occupied conformers of integrin αIIbβ3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function.
J Biol Chem
.
1991
;
266
:
17106
-17111.
70
Andre P, Prasad KS, Denis CV, et al. CD40L stabilizes arterial thrombi by a beta3 integrin-dependent mechanism.
Nat Med
.
2002
;
8
:
247
-252.
71
Peterson JA, Nyree CE, Newman PJ, Aster RH. A site involving the “hybrid” and PSI homology domains of GPIIIa (β3 integrin subunit) is a common target for antibodies associated with quinine-induced immune thrombocytopenia.
Blood
.
2002
;
101
:
937
-942.
72
Bougie DW, Wilker PR, Wuitschick ED, et al. Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa.
Blood
.
2002
;
100
:
2071
-2076.
73
Kamata T, Ambo H, Puzon-McLaughlin W, et al. Critical cysteine residues for regulation of integrin αIIbβ3 are clustered in the epidermal growth factor domains of the β3 subunit.
Biochem J
.
2004
;
378
:
1079
-1082.
74
Essex DW, Chen K, Swiatkowska M. Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane.
Blood
.
1995
;
86
:
2168
-2173.
75
O'Neill S, Robinson A, Deering A, Ryan M, Fitzgerald DJ, Moran N. The platelet integrin αIIbβ3 has an endogenous thiol isomerase activity.
J Biol Chem
.
2000
;
275
:
36984
-36990.
76
Yan B, Smith JW. A redox site involved in integrin activation.
J Biol Chem
.
2000
;
275
:
39964
-39972.
77
Yan BX, Smith JW. Mechanism of integrin activation by disulfide bond reduction.
Biochemistry
.
2001
;
40
:
8861
-8867.
78
Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands.
Trends Cell Biol
.
2001
;
11
:
130
-135.
79
Lagadec P, Dejoux O, Ticchioni M, et al. Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow.
Blood
.
2003
;
101
:
4836
-4843.
80
Fujimoto TT, Katsutani S, Shimomura T, Fujimura K. Thrombospondin-bound integrin associated protein (CD47) physically and functionally modifies integrin αIIbβ3 by its extracellular domain.
J Biol Chem
.
2003
;
278
:
26655
-26665.
81
Critchley DR. Focal adhesions: the cytoskeletal connection.
Curr Opin Cell Biol
.
2000
;
12
:
133
-139.
82
Calderwood DA, Yan B, de Pereda JM, et al. The phosphotyrosine binding-like domain of talin activates integrins.
J Biol Chem
.
2002
;
277
:
21749
-21758.
83
Calderwood DA. Integrin activation.
J Cell Sci
.
2004
;
117
:
657
-666.
84
Ling K, Doughman RL, Firestone AJ, Bunce MW, Anderson RA. Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal adhesions.
Nature
.
2002
;
420
:
89
-93.
85
Patil S, Jedsadayanmata A, Wencel-Drake JD, Wang W, Knezevic I, Lam SC. Identification of a talin-binding site in the integrin β3 subunit distinct from the NPLY regulatory motif of post-ligand binding functions: the talin n-terminal head domain interacts with the membrane-proximal region of the β3 cytoplasmic tail.
J Biol Chem
.
1999
;
274
:
28575
-28583.
86
Calderwood DA, Zent R, Grant R, Rees DJG, Hynes RO, Ginsberg MH. The talin head domain binds to integrin β subunit cytoplasmic tails and regulates integrin activation.
J Biol Chem
.
1999
;
274
:
28071
-28074.
87
Calderwood DA, Ginsberg MH. Talin forges the links between integrins and actin.
Nat Cell Biol
.
2003
;
5
:
694
-697.
88
Tadokoro S, Shattil SJ, Tai V, et al. Talin binding to integrin β cytoplasmic tails: a final common step in integrin activation.
Science
.
2003
;
302
:
103
-106.
89
Ulmer TS, Calderwood DA, Ginsberg MH, Campbell ID. Domain-specific interactions of talin with the membrane-proximal region of the integrin β3 subunit.
Biochemistry
.
2003
;
42
:
8307
-8312.
90
Garcia-Alvarez B, de Pereda JM, Calderwood DA, et al. Structural determinants of integrin recognition by talin.
Mol Cell
.
2003
;
11
:
49
-58.
91
Wang R, Shattil SJ, Ambruso DR, Newman PJ. Truncation of the cytoplasmic domain of β3ina variant form of Glanzmann thrombasthenia abrogates signaling through the integrin αIIbβ3 complex.
J Clin Invest
.
1997
;
100
:
2393
-2403.
92
Bertagnolli ME, Beckerle MC. Regulated membrane-cytoskeleton linkages in platelets.
Ann NY Acad Sci
.
1994
;
714
:
88
-100.
93
Martel V, Racaud-Sultan C, Dupe S, et al. Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides.
J Biol Chem
.
2001
;
276
:
21217
-21227.
94
Tapley P, Howwitz AF, Buck CA, Duggan K, Rohrschneider L. Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts.
Oncogene
.
1989
;
4
:
325
-333.
95
Yan B, Calderwood DA, Yaspan B, Ginsberg MH. Calpain cleavage promotes talin binding to the β3 integrin cytoplasmic domain.
J Biol Chem
.
2001
;
276
:
28164
-28170.
96
Ling K, Doughman RL, Iyer VV, et al. Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch.
J Cell Biol
.
2003
;
163
:
1339
-1349.
97
Brown NH, Gregory SL, Rickoll WL, et al. Talin is essential for integrin function in Drosophila.
Dev Cell
.
2002
;
3
:
569
-579.
98
Jiang G, Giannone G, Critchley DR, Fukumoto E, Sheetz MP. Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin.
Nature
.
2003
;
424
:
334
-337.
99
Barry WT, Boudignon-Proudhon C, Shock DD, et al. Molecular basis of CIB binding to the integrin αIIb cytoplasmic domain.
J Biol Chem
.
2002
;
277
:
28877
-28883.
100
Tsuboi S. Calcium integrin-binding protein activates platelet integrin αIIbβ3.
J Biol Chem
.
2002
;
277
:
1919
-1923.
101
Buensuceso C, Arias-Salgado EG, Shattil SJ. Protein-protein interactions in platelet αIIbβ3 signaling.
Semin Thromb Hemost
. In press.
102
Prevost N, Woulfe D, Tanaka T, Brass LF. Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred.
Proc Natl Acad Sci U S A
.
2002
;
99
:
9219
-9224.
103
Angelillo-Scherrer A, De Frutos PG, Aparicio C, et al. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.
Nature Med
.
2001
;
7
:
215
-221.
104
Prevost N, Woulfe D, Tognolini M, Brass LF. Contact-dependent signaling during the late events of platelet activation.
J Thromb Haemost
.
2003
;
1
:
1613
-1627.
105
Phillips DR, Prasad KS, Manganello J, Bao M, Nannizzi-Alaimo L. Integrin tyrosine phosphorylation in platelet signaling.
Curr Opin Cell Biol
.
2001
;
13
:
546
-554.
106
Hartwig JH, Kung S, Kovacsovics T, et al. 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
.
1996
;
271
:
32986
-32993.
107
Leng L, Kashiwagi H, Ren X-D, Shattil SJ. RhoA and the function of platelet integrin αIIbβ3.
Blood
.
1998
;
91
:
4206
-4215.
108
Yuan YP, Kulkarni S, Ulsemer P, et al. The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells.
J Biol Chem
.
1999
;
274
:
36241
-36251.
109
Bearer EL, Prakash JM, Li Z. Actin dynamics in platelets.
Int Rev Cytol
.
2002
;
217
:
137
-182.
110
Azim AC, Barkalow K, Chou J, Hartwig JH. Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor.
Blood
.
2000
;
95
:
959
-964.
111
Hartwig JH, Barkalow K, Azim A, Italiano J. The elegant platelet: signals controlling actin assembly.
Thromb Haemost
.
1999
;
82
:
392
-398.
112
Obergfell A, Eto K, Mocsai A, et al. Coordinate interactions of Csk, Src, and Syk kinases with αIIbβ3 initiate integrin signaling to the cytoskeleton.
J Cell Biol
.
2002
;
157
:
265
-275.
113
Wonerow P, Pearce AC, Vaux DJ, Watson SP. A critical role for phospholipase Cgamma2 in αIIbβ3-mediated platelet spreading.
J Biol Chem
.
2003
;
278
:
37520
-37529.
114
Hantgan RR, Lyles DS, Mallett TC, Rocco M, Nagaswami C, Weisel JW. Ligand binding promotes the entropy-driven oligomerization of integrin αIIbβ3.
J Biol Chem
.
2003
;
278
:
3417
-3426.
115
Prasad KS, Andre P, He M, Bao M, Manganello J, Phillips DR. Soluble CD40 ligand induces β3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling.
Proc Natl Acad Sci U S A
.
2003
;
100
:
12367
-12371.
116
de Virgilio M, Kiosses WB, Shattil SJ. Proximal, selective and dynamic interactions between integrin αIIbβ3 and protein tyrosine kinases in living cells.
J Cell Biol
.
2004
;
165
:
305
-311.
117
Woodside DG, Obergfell A, Leng L, et al. Activation of Syk protein tyrosine kinase mediated by interaction with integrin β cytoplasmic domains.
Curr Biol
.
2001
;
11
:
1799
-1804.
118
Miranti C, Leng L, Maschberger P, Brugge JS, Shattil SJ. Integrin-induced assembly of a Sykand Vav1-regulated signaling pathway independent of actin polymerization.
Curr Biol
.
1998
;
8
:
1289
-1299.
119
Judd BA, Myung PS, Leng L, et al. Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through αIIbβ3 and collagen receptors.
Proc Natl Acad Sci. U S A
.
2000
;
97
:
12056
-12061.
120
Obergfell A, Judd BA, del Pozo MA, Schwartz MA, Koretzky G, Shattil S. The molecular adapter SLP-76 relays signals from platelet integrin αIIbβ3 to the actin cytoskeleton.
J Biol Chem
.
2001
;
276
:
5916
-5923.
121
Jenkins AL, Nannizzi-Alaimo L, Silver D, et al. Tyrosine phosphorylation of the β3 cytoplasmic domain mediates integrin-cytoskeletal interactions.
J Biol Chem
.
1998
;
273
:
13878
-13885.
122
Cowan KJ, Law DA, Phillips DR. Identification of Shc as the primary protein binding to the tyrosinephosphorylated β3 subunit of αIIbβ3 during outside-in integrin platelet signaling.
J Biol Chem
.
2000
;
275
:
36423
-36429.
123
Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in αIIbβ3 signalling and platelet function.
Nature
.
1999
;
401
:
808
-811.
124
Calderwood DA, Shattil SJ, Ginsberg MH. Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling.
J Biol Chem
.
2000
;
275
:
22607
-22610.
125
Pavalko FM, Chen NX, Turner CH, et al. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton integrin interactions.
Am J Physiol
.
1998
;
275
:
C1591
-C1601.
126
Izaguirre G, Aguirre L, Hu YP, et al. The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.
J Biol Chem
.
2001
;
276
:
28676
-28685.
127
Reddy KB, Bialkowska K, Fox JEB. Dynamic modulation of cytoskeletal proteins linking integrins to signaling complexes in spreading cells: role of skelemin in initial integrin-induced spreading.
J Biol Chem
.
2001
;
276
:
28300
-28308.
128
Wu CY, Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.
J Cell Biol
.
2001
;
155
:
505
-510.
129
Naik MU, Naik UP. Calcium- and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen.
Blood
.
2003
;
102
:
3629
-3636.
130
Parsons JT. Focal adhesion kinase: the first ten years.
J Cell Sci
.
2003
;
116
:
1409
-1416.
131
Helluin O, Chan C, Vilaire G, Mousa S, DeGrado WF, Bennett JS. The activation state of αVβ3 regulates platelet and lymphocyte adhesion to intact and thrombin-cleaved osteopontin.
J Biol Chem
.
2000
;
275
:
18337
-18343.
132
Jung SM, Moroi M. Platelet collagen receptor integrin α2β1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.
Eur J Biochem
.
2001
;
268
:
3513
-3522.
133
Schoolmeester A, Vanhoorelbeke K, Katsutani S, et al. Monoclonal antibody IAC-1 is specific for activated α2β1 and binds to amino acid 199-201 of the integrin α2 I-domain.
Blood
.
2004
;
104
:
390
-396.
134
Rao AK, Gabbeta J. Congenital disorders of platelet signal transduction.
Arterioscler Thromb Vasc Biol
.
2000
;
20
:
285
-289.
135
Cattaneo M. Inherited platelet-based bleeding disorders.
J Thromb Haemost
.
2003
;
1
:
1628
-1636.
136
Emambokus NR, Frampton J. The glycoprotein IIb molecule is expressed on early murine hematopoietic progenitors and regulates their numbers in sites of hematopoiesis.
Immunity
.
2003
;
19
:
33
-45.
137
Weinreb PH, Simon KJ, Rayhorn P, et al. Function-blocking integrin αVβ6 monoclonal antibodies: distinct ligand-mimetic and nonligandmimetic classes.
J Biol Chem
.
2004
;
279
:
17875
-17887.
138
Ni H, Yuen PS, Papalia JM, et al. Plasma fi-bronectin promotes thrombus growth and stability in injured arterioles.
Proc Natl Acad Sci U S A
.
2003
;
100
:
2415
-2419.
139
Newman PJ. Platelet GPIIb-IIIa: molecular variations and alloantigens.
Thromb Haemost
.
1991
;
66
:
111
-118.
140
Harrison SC. Variation on a Src-like theme.
Cell
.
2003
;
112
:
737
-740.
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