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Platelet membrane glycoproteins mediate most of the interactions between platelets and their external environment. Receptors can receive signals from outside the platelet and transmit signals inside. In addition, glycoprotein receptors receive signals from inside the platelet that affect their external domain functions. Platelet glycoprotein receptors are grouped into several different receptor families (integrins, leucine-rich glycoproteins, immunoglobulin cell adhesion molecules, selectins, tetraspanins, and seven-transmembrane domain receptors; see Table 112–4). One member of the integrin family, integrin αIIbβ3, is virtually unique to platelets (and their precursors, megakaryocytes), whereas the leucine-rich glycoproteins GPIb/IX and GPV appear to have highly restricted but not uniquely platelet expression patterns, including cytokine-activated endothelial cells.801,802 All of the other receptors are expressed more widely on other cell types.
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Integrin receptors are heterodimeric complexes composed of an α subunit containing three or four divalent cation binding domains and a β subunit rich in disulfide bonds. Both subunits are transmembrane glycoproteins and are coded by different genes. There are at least 18 α subunits and eight β subunits.43,803,804 Three major families of integrin receptors are recognized based on the β subunit: β1, β2, and β3. Integrins are widely distributed on different cell types, and each integrin demonstrates unique ligand-binding properties. Integrin receptors mediate interactions between cells and proteins or proteins on cells; they are also involved in protein trafficking in cells. Integrin receptors can also transduce messages from outside the cell to inside the cell, and from inside the cell to outside the cell.
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Integrin αIIbβ3 (Also Termed GPIIb/IIIa, Fibrinogen Receptor, and CD41/CD61)
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The integrin αIIbβ3 complex, a member of the β3 integrin receptor family, is the dominant platelet receptor, with 80,000 to 100,000 receptors present on the surface of a resting platelet (Fig. 112–11).805,806,807,808,809,810,811,812 Another 20,000 to 40,000 receptors are present inside platelets, primarily in α-granule membranes, but also in dense bodies and membranes lining the open canalicular system; these receptors are able to join the plasma membrane when platelets are activated and undergo the release reaction.813,814,815 On average, integrin αIIbβ3 receptors are less than 20 nm apart on the platelet surface and thus are among the most densely expressed adhesion/aggregation receptors present on any cell type.
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On resting platelets, integrin αIIbβ3 has low affinity for fibrinogen in solution, but when platelets are activated with ADP, epinephrine, thrombin, or other agonists, integrin αIIbβ3 binds fibrinogen relatively strongly.808,816 Activation induces changes in the integrin αIIbβ3 receptor itself that are responsible for the change in fibrinogen-binding affinity, but changes in the microenvironment surrounding integrin αIIbβ3 may also be involved. The integrin αIIbβ3 receptors in α granules appear to cycle to and from the plasma membrane.817 This recycling helps to explain the ability of the integrin to take up fibrinogen from plasma and transport it to α granules, where it is concentrated.375,818
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Data from other integrin receptors identified a cell recognition sequence composed of RGD in the ligand fibronectin,819,820 and this same sequence is important in ligand binding to integrins αVβ3 and αIIbβ3. Fibrinogen contains one RGD sequence near the carboxy terminus of each of the two Aα chains (amino acids 572 to 574) and another at amino acids 95 to 97.821 In addition, the carboxyterminal 12 amino acid region of each of the two γ chains (amino acids 400 to 411) contains a sequence that includes Lys-Gln-Ala-Gly-Asp-Val, which is the most important in the binding of fibrinogen to platelets.822,823,824,825,826 VWF contains an RGD sequence in its carboxyterminal domain and that region mediates the binding to integrin αIIbβ3.809,810,812 Small, synthetic peptides containing the RGD or γ-chain sequence inhibit the binding of fibrinogen to platelets, and these observations have been exploited to produce therapeutic agents (tirofiban and eptifibatide) to inhibit platelet thrombus formation827 (Chap. 134). Similarly, monoclonal antibodies that inhibit binding of ligands to integrin αIIbβ3 have been developed and a mouse/human chimeric Fab fragment of one of them has been developed into a drug (abciximab) that is an effective antiplatelet agent.
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The binding of fibrinogen to integrin αIIbβ3 appears to be a multistep process808,828,829,830,831,832,833: (1) the initial interaction is most likely via the γ-chain carboxyterminal region(s) and divalent cation-dependent823,824,825,826; (2) subsequent interactions enhance the binding and internalization of the fibrinogen834 and render it irreversible, even when divalent cations are removed835; (3) binding of fibrinogen induces changes in the receptor that can be recognized by antibodies (ligand-induced binding sites [LIBSs])442,826; (4) binding of fibrinogen to integrin αIIbβ3 induces changes in fibrinogen (receptor-induced binding sites) that can be recognized by antibodies and may involve exposure of the Aα chain Arg-Gly-Asp-Phe sequence at amino acids 95 to 98836,837; and (5) fibrinogen binding induces receptor clustering.251,838
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By electron microscopy, the receptors have a globular head of 8 × 12 nm and two 18-nm long tails representing the carboxyterminal regions of each subunit, including their hydrophobic transmembrane domains.839,840 Crystallographic, electron microscopic, electron and neutron scattering, and biochemical data from integrin αIIbβ3 and the related integrin αVβ3 receptor indicate that the unactivated receptors are in a bent conformation and that activation involves both extension of the receptor head and a swing out motion in the β3 subunit.149,827,841–853 A three-dimensional reconstruction of integrin αIIbβ3 in a lipid bilayer nano disc from negative-stain electron microscopy images supports a compact conformation of the inactive receptor, but unlike the crystal structure of the ectodomain, the legs are not parallel and straight.848
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Integrin αIIbβ3 shares the same basic structural features of all integrin receptors (Table 112–4).30,848 The α subunit, αIIb, is a transmembrane protein with four characteristic divalent cation-binding sites (see Fig. 112–11). The mature protein contains 1008 amino acids43,854 with one transmembrane domain; during processing, it is cleaved into a heavy chain and a light chain connected by a disulfide bond. The β subunit, β3, contains 762 amino acids and is rich in cysteine residues, with a characteristic cysteine-rich region near its transmembrane domain.43,855 The integrin αIIb and β3 cytoplasmic tails consist of 20 and 47 amino acids, respectively. The genes coding for αIIb and β3 are very close to each other on chromosome 17 at q21.32, but are not so close as to share common regulatory domains.856,857 Both proteins are synthesized in megakaryocytes and join to form a calcium-dependent, noncovalent complex in the rough endoplasmic reticulum.858 Calnexin probably serves as a chaperone for integrin αIIb,859 but it is unclear which chaperone(s) are involved in integrin β3 folding and/or integrin αIIbβ3 complex formation. The integrin αIIbβ3 complex subsequently undergoes further processing in the Golgi apparatus, where the carbohydrate structures undergo maturation and the pro-GPIIb molecule is cleaved into its heavy and light chains by furin or a similar enzyme.860,861 Approximately 15 percent of the mass of both integrins αIIb and β3 are composed of carbohydrate.862 The mature integrin αIIbβ3 complex is then transported to the plasma membrane or the membranes of α granules or dense bodies. If integrins αIIb and β3 do not form a proper complex, either because of a structural abnormality in either subunit or the failure to synthesize one of the subunits, the subunit(s) that are synthesized are rapidly degraded and so are not expressed on the membrane surface (Chap. 121). Degradation of integrin αIIb appears to involve retro-translocation from the endoplasmic reticulum into the cytoplasm, ubiquitination, and proteolysis by the megakaryocyte proteasome.859
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Both integrins αIIb and β3 are composed of a series of domains (see Fig. 112–11). The aminoterminal region of integrin αIIb contains a seven-blade β-propeller domain, and each blade is composed of four β strands connected by loops. The propeller interacts with the βA (I-like) domain of integrin β3, forming the globular head region observed in electron micrographs. The four calcium ions bound by the propeller domain interact with β hairpin loops in blades four to seven that extend away from the interface with integrin β3. In addition, there is a unique integrin αIIb cap subdomain made up of four loops from blades one to three that are unique to αIIb and contribute to its ligand binding specificity. The remainder of the extracellular components of integrin αIIb are made up of a thigh, genu (knee-like), and two calf domains,250 much like the structure of the related integrin αV subunit.841,844 The cytoplasmic domain of integrin αIIb interacts with the cytoplasmic domain of integrin β3 and the interaction is important in controlling activation of the holoreceptor.863,864,865,866 The cytoplasmic domain of integrin αIIb has a GFFKR sequence near the membrane that is thought to control inside-out activation of the integrin receptors because mutations or deletions in this region result in the receptor adopting a conformation with high affinity for fibrinogen.867,868,869,870,871 A number of studies using mutagenesis and nuclear magnetic resonance (NMR) identified different structures for the transmembrane and cytoplasmic domains, and differences in the relative roles of heterodimeric and homodimeric associations.864,872,873,874,875 Disrupting the conformation of this region also results in a constitutively high-affinity receptor,876,877 which has led to the conclusion that inside-out activation of integrin αIIbβ3 requires separation of the transmembrane and cytoplasmic domains, but it remains possible that more subtle changes in the cytoplasmic and transmembrane domains may be sufficient.848
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The integrin β3 subunit domains are not linearly arranged because the first domain (PSI [plexins, semaphorins, and integrins]) was subjected to the insertion of a hybrid domain, which itself was subjected to the insertion of a βA (I-like) domain; the latter domain is homologous to the VWF A domain and integrin I domains, both of which bind ligands (see Fig. 112–11).827,878 The double insertion in the PSI domain explains why there is a “long range” disulfide bond extending from C13 to C435; thus, even though the βA domain makes contact with the integrin αIIb propeller (via Arg261 and other residues that interact with two rings of hydrophobic residues in the integrin αIIb “cage”), it is not the aminoterminus of the molecule. The PSI domain contains Leu33, which defines the PlA1 (HPA-1a) specificity, as opposed to the alloantigen PlA2 (HPA-1b), which is produced by a Pro33 polymorphism (Chap. 137). The integrin β3 leg is composed of four integrin EGF domains that are rich in disulfide bonds. In the crystal structure, this region interacts with the integrin αIIb stalk region and the globular head in the bent, unactivated receptor, but these interactions are less prominent in the three-dimensional reconstruction of the inactive receptor not in the activated receptor.250,827,848 Mutations in the integrin EGF domains, including cysteine residues, can activate the receptor as can the binding of monoclonal antibodies.879,880,881,882 The importance of the normal disulfide bond pairings in integrin β3 is further supported by data demonstrating that certain reducing agents can cause activation of integrin αIIbβ3, fibrinogen binding, and platelet aggregation,883,884 and an enzyme capable of catalyzing the exchange of thiol groups and disulfide in proteins (PDI) has been identified on the surface of platelets and in platelet releasates.883,885,886,887 Thiol-disulfide exchange in integrins αIIbβ3 and αVβ3 is implicated as a contributor to clot retraction.888 Moreover, regions in integrin β3 itself have the same consensus sequence (CGXC) present in PDI that is thought to mediate the catalysis.889 One model suggests that integrin αIIbβ3 can achieve a low level of activation without alterations in disulfide bonds, but that maximal activation requires PDI or similar activity along with a source of thiols such as plasma glutathione or a membrane NAD(P)H oxidoreductase system.883 Inhibition of PDI and other enzymes that mediate thiol-disulfide exchange (ERp57, ERp5) reduces platelet thrombus formation.890,891 It is still unclear, however, whether disulfide bond alterations contribute to activation in vivo under physiologic or pathologic conditions.
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Transmembrane domain structures of integrin αIIb and integrin β3 have been proposed based on NMR and structural modeling studies.871,873,874,892,893,894,895,896 Because the integrin αIIb transmembrane helix is shorter than the integrin β3 helix, they traverse the membrane at an angle of approximately 25 degrees. The association of the integrin αIIb and integrin β3 ectodomains near the site of entry into the membrane results in the transmembrane helices being directly juxtaposed in the region of the membrane closest to the ectodomain (outer membrane clasp). Near the cytoplasmic end of the membrane the helices are held together by an inner membrane clasp composed of the integrin αIIb residues immediately after the end of the helix (GFFKR), with the membrane reimmersion of F992 and F993 filling the gap and interacting with integrin β3 W715 and I719, with integrin αIIb R995 creating a salt bridge with integrin β3 723 and perhaps residue 726.897,898 Of note, these regions are conserved in many other integrins receptors and so the basic mechanism may be common to many of the receptors.
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Inside-out signaling is accomplished by the talin F3 domain binding to the integrin β3 cytoplasmic domain, which is proposed to disrupt the inner membrane clasp.34,244,245,863,865,866,869,870,872,876,892,899,900 This may be facilitated by migfilin displacing filamin from the integrin β3 cytoplasmic domain as the latter interaction may prevent talin binding.901 Talin binding results in dissociation of the transmembrane helices and reorganization of the cytoplasmic region of integrin β3 into a more extended helix. Integrin αIIbβ3 ectodomain chain separation, headpiece extension, and integrin β3 swing out then follow, either spontaneously or as a result of the traction force generated by the cytoskeleton on integrin β3 through talin.149 Outside-in signaling is presumed to be initiated by loss of ectodomain interactions between the membrane-proximal regions of integrins αIIb and β3, perhaps as a result of ligand binding producing even greater integrin β3 swing out, resulting in disruption of the outer membrane clasp and subsequent dissociation of the transmembrane helices. This potentially may facilitate the interaction of the cytoplasmic domains with cytoskeletal elements and signaling molecules.
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The integrin β3 tail also contains two NXXY motifs and Y747 and Y759 within one of these motifs are phosphorylated upon platelet aggregation, thus producing docking sites for signaling molecules.235 Studies in mice and in recombinant systems demonstrate a role for the sites in clot retraction and platelet aggregate stability.291,902
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A number of proteins have been shown to bind to the cytoplasmic domains of integrin αIIb and/or β3, either directly or through interactions with other proteins, including signaling molecules (Src, Shc, FAK, paxillin, and ILK, all of which bind to integrin β3), cytoskeletal proteins (kindlin-3, skelemin, α-actin, and myosin, which bind to integrin β3, and filamin and talin, which bind to integrins αIIb and/or β3), and other proteins (β3-endonexin and CD98, which bind to integrin β3, and CIB and calreticulin, which bind to αIIb) (Fig. 112–12).244,866,903–919 These interactions are important in mediating inside-out signaling and outside-in signaling.235 JAM-A is a negative regulator of outside-in activation by integrin αIIbβ3 that acts by regulating activation of Src.920 Similarly, PECAM-1 serves as an inhibitor of integrin αIIbβ3 activation through a sequential phosphorylation mechanism.921,922 Force on the integrin β3 cytoplasmic domain by actin–myosin action may supply the energy for the conformational change in integrin αIIbβ3 from bent to extended.250
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The junction between the integrin αIIb propeller and the β3 βA (I-like) domain is the site of ligand binding to integrin αIIbβ3 (see Fig. 112–11). This region of integrin β3 contains three divalent cation binding sites: MIDAS (metal ion-dependent adhesion site), ADMIDAS (adjacent to MIDAS), and SyMBS (synergy metal binding site).250 The latter was previously termed the ligand-associated metal binding site (LIMBS) based on the crystal structure of integrin αVβ3.844,845
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The crystal structure of integrin αVβ3 demonstrated that an RGD peptide bound primarily via interactions between the Arg in the peptide and two Asp residues (D150 and D218) in integrin αV and between the Asp in the peptide and the MIDAS cation.845 The binding pocket in integrin αIIbβ3 is similar but differs in that only one Asp in integrin αIIb (D224) is available to interact with an Arg (or Lys as in the fibrinogen γ-chain peptide), the distance between D224 in integrin αIIb and the MIDAS cation is longer, and a cap subdomain of the integrin αIIb propeller contributes Phe160 to a hydrophobic exosite in combination with Tyr190.149,827 As a result, the pocket is able to accommodate the longer fibrinogen γ-chain C-terminal peptide better, with the peptide’s Asp and C-terminal Val carboxyls interacting with the MIDAS and ADMIDAS cations, respectively.826 It also explains why integrin αIIbβ3 can bind peptides containing the longer Lys residue (KGD peptides).923 Crystal structures are also available for the integrin αIIbβ3 receptor with the drugs eptifibatide and tirofiban, which are effective antithrombotic agents because of their ability to block ligand binding to integrin αIIbβ3, and demonstrate specificity for integrin αIIbβ3 compared to integrin αVβ3.827 The basis of the specificity of these agents involves in part their interaction with the integrin αIIb-specific exosite and the greater length between their positive and negative charges.827 The third integrin αIIbβ3 antagonist drug, abciximab, is a chimeric murine monoclonal antibody Fab fragment. Its epitope has been localized to a region on integrin β3 very close to the MIDAS, suggesting that it works by steric interference with ligand binding, disruption of the binding pocket, or both mechanisms.
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Two major conformational changes in integrin αIIbβ3 have been described in association with activation: headpiece extension and integrin β3 hybrid and PSI domain swing-out (see Fig. 112–11).250,827,853 Headpiece extension can contribute to ligand binding by enhancing access to the binding site; it can also contribute to platelet aggregation by extending the receptor out further from the platelet surface,924 thus facilitating the ability of fibrinogen to bridge between platelets.846 The integrin β3 hybrid and PSI domain swing-out motion appears to enhance ligand binding, but the precise mechanism is unclear.826,847,850 Swing-out is associated with movement of the ADMIDAS metal ion and the α1-β1 loop toward the MIDAS with the latter movement stabilized by the interaction of two backbone nitrogens in the α1-β1 loop with the ligand carboxyl oxygen, thus reinforcing the binding to the MIDAS metal ion.149,826 Mutations that produce swing-out of the hybrid and PSI domains result in constitutive ligand binding to integrin αIIbβ3.925
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Binding of fibrinogen to platelet integrin αIIbβ3 leads to platelet aggregation, presumably via crosslinking of integrin molecules on two different platelets by fibrinogen.840 The dimeric and relatively rigid structure of fibrinogen, and the location of the binding sites at the ends of the γ chains are all consistent with such a model as the two binding sites on a single fibrinogen molecule are probably more than 45 nm apart. Soon after fibrinogen binds, it can be dissociated from the platelet by chelating the divalent cations, but the binding becomes irreversible within an hour.835 Fibrinogen binding alone is not sufficient for platelet aggregation, but the events necessary after fibrinogen binding, which probably include ligand- and/or cytoskeletal-mediated receptor clustering, are not well understood.95,835,926,927 After ligands bind to integrin αIIbβ3, “outside-in” signaling through the integrin can occur, resulting in a number of phosphorylation events, changes in the platelet cytoskeleton, platelet spreading, and even initiation of protein translation.236,237,928
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In addition to fibrinogen, several other proteins can bind to integrin αIIbβ3 on activated platelets, including VWF, fibronectin, vitronectin, thrombospondin, and prothrombin390,675,929; each of these contains an RGD sequence in the region implicated in the initial interaction with platelets. There are subtle differences in the binding of each of these ligands, however, with regard to divalent cation preference and competent activating agents. The binding of all of these other ligands can also be inhibited by RGD-containing peptides, indicating a common requirement for the interaction between the RGD sequence in the protein and the RGD-binding site in integrin αIIbβ3.930,931
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Platelet aggregation measured in the aggregometer ex vivo depends upon fibrinogen binding to integrin αIIbβ3. It is less clear whether fibrinogen is the most important ligand supporting platelet aggregation in vivo since studies performed in model systems under flowing conditions indicate that VWF is the major ligand at higher shear rates.932 Even in the aggregometer, VWF can partially substitute for fibrinogen if the fibrinogen concentration is very low.933 In vivo, mice deficient in both VWF and fibrinogen still make platelet thrombi in response to vascular injury.934,935,936 Although fibronectin was initially implicated in supporting the development of such thrombi, mice deficient in fibrinogen, VWF, and fibronectin have paradoxically increased platelet aggregation and thrombus formation, suggesting that fibronectin may play an inhibiting role in thrombus formation under certain circumstances.373
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Although resting platelets do not bind soluble fibrinogen (or other adhesive glycoproteins) to an appreciable extent, they can adhere to fibrinogen immobilized on a surface.825,937 This activation-independent adhesion may be from alterations in the structure of fibrinogen when it is immobilized on a surface.836,938 Alternatively, there may always be a few integrin αIIbβ3 receptors that are transiently in the proper conformation to bind fibrinogen, and immobilization may result in high local density of fibrinogen and favorable kinetics for adhesion. Finally, it is possible that even low-affinity fibrinogen interactions with integrin αIIbβ3 are sufficient to initiate integrin interactions with the cytoskeleton such that actin-myosin-induced contraction provides the energy required for the conformational changes needed to achieve higher affinity binding.250
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Fibrinogen and/or fibrin have been identified on the surface of damaged blood vessels; thus it is possible that integrin αIIbβ3 mediates platelet adhesion under those circumstances.939 In contrast, integrin αIIbβ3 on resting platelets does not appear to be able to mediate adhesion to VWF or fibronectin940; if platelets are activated, however, integrin αIIbβ3 can support adhesion to these glycoproteins.930 In models of platelet accumulation under flowing conditions, αIIbβ3 acts in synergy with GPIb/IX, VWF, and fibrinogen at the apex of thrombi, where shear forces are greatest.28,941,942 The integrin αIIbβ3 has also been implicated in platelet spreading after adhesion,227,228,943 and it is necessary for clot retraction (see above) and the uptake of plasma fibrinogen into platelet α granules.818,944
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Less-well-defined roles for integrin αIIbβ3 have been suggested in the binding of plasminogen,688 calcium transport across the platelet membrane,945,946,947 IgE binding to platelets leading to parasite cytotoxicity,948 and interactions with the Borrelia species spirochetes that cause Lyme disease949 and hantavirus.950 Integrin αIIbβ3 also mediates factor XIIIa binding to platelets, but this is primarily as a result of factor XIII’s association with fibrinogen.456 Factor XIIIa and calpain have also been implicated in limiting platelet–platelet interactions after activation by adhesion to collagen.951
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Integrin α2β1 (Also Termed GPIa/IIa, Collagen Receptor, VLA-2 and CD49b/CD29)
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Integrin α2β1 (GPIa/IIa) is widely distributed on different cell types and can mediate adhesion to collagen.19,20,952,953,954,955,956,957 The integrin α2 subunit (GPIa) contains a region of 220 amino acids inserted in the aminoterminal β-propeller region (I domain) that is homologous to similar regions in other proteins that are known to interact with collagen, including VWF and cartilage matrix protein.958 This region has a MIDAS and crystallographic data of the α2 I domain in complex with a CRP containing the type I collagen sequence GFOGER (where O indicates hydroxyproline) demonstrated that the glutamic acid in the peptide coordinates Mg2+ binding in the MIDAS.959,960,961 The integrin α2β1 I domain can assume a variety of conformations, going from inactive (closed), through intermediate or low affinity, to active high affinity.952,962
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Both integrin α2β1 and GPVI appear to participate in platelet interactions with collagen.963,964,965 Bleeding defects have been described in patients with decreased levels of integrin α2β1 and GPVI, but the precise contributions of the decreases in these receptors is uncertain (Chap. 121). Although integrin α2β1 is capable of supporting adhesion to collagen without exogenous activators, like integrin αIIbβ3, it appears to be able to increase its affinity for ligand in response to inside-out activation.966,967 Potential initiators of integrin α2β1 activation include signaling after GPVI interaction with collagen and GPIb-mediated adhesion to VWF, perhaps acting via actin polymerization.959,968,969,970 Thus, one possible scenario is that following GPIb-mediated adhesion to VWF and collagen adhesion and activation mediated by GPVI, integrin α2β1 may promote firm adhesion to collagen, stabilize thrombus growth on collagen, and promote procoagulant activity.971,972 In addition, the affinity of integrin α2β1 may also be modulated by alterations in disulfide bonds since inhibition of platelet PDI and sulfhydryl blocking agents inhibit integrin α2β1-mediated platelet adhesion to type I collagen and to the related peptide GFOGER.883,973 The state of the collagen may also influence whether integrin α2β1 or GPVI mediates the interaction with collagen, because GPVI appears to mediate adhesion to fibrillar collagen whereas integrin α2β1 preferentially adheres to collagen that has been treated with partial protease digestion.28,974
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Ligand binding to integrin α2β1 is enhanced in the presence of magnesium or manganese and is inhibited by calcium, and thus the conditions in human blood, where calcium concentrations are higher than those of magnesium, do not provide optimal cation concentrations for the receptor’s function.975 Integrin α2β1 can, however, mediate platelet adhesion to collagen in heparinized blood956,975 and inhibitors of integrin α2β1 inhibit thrombus formation in animal models.976,977,978 Regions of collagen type I have been implicated as potential binding sites for integrin α2β1979; the peptide sequence 502 to 516 of collagen type I α1 chain, which contains a Gly-Glu-Arg (GER) sequence, may be of particular importance,980 but other regions of the collagen molecule may also be important.981 In type III collagen, amino acids 522 to 528 of fragment α1 (III) CB4 contains a binding region for α2β1.982
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Three different alleles for the integrin α2 gene, which differ at nucleotides 807 (T or C) and 1648 (G or A), have been described.983 The 807 substitution does not affect the amino acid sequence, but the 1648 substitution causes a change from Glu to Lys, resulting in the Brb and Bra alloantigens (HPA-5a and HPA-5b) (Chap. 137). Allele 1 (T-G) is present in 39 percent of individuals, allele 2 (C-G) in 53 percent, and allele 3 (C-A) in 7 percent.984,985 Individuals with allele 1 have higher integrin α2β1 platelet density than individuals with allele 2, and individuals with allele 3 have the lowest density; the density of integrin α2β1correlates with platelet deposition on collagen under flow. The association of these polymorphisms with cardiovascular disease morbidity and mortality, including the risk of developing MI986,987 and stroke,988 has been extensively study without firm conclusions, although there is some suggestion that they may be associated with cardiovascular risk.983,989,990,991,992
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Integrin α2β1 is probably linked to the membrane skeleton.993 Its ligand specificity appears to be determined by the cell on which it is expressed, since on endothelial cells it functions as a laminin receptor as well as a collagen receptor.994,995 Engagement of integrin α2β1 is capable of initiating platelet protein synthesis.236 Integrin α2β1 has been implicated in megakaryocyte development and platelet formation. In particular, loss of activated integrin α2β1 receptors on the surface of megakaryocytes, as a result of interacting with collagen, has been implicated in the transition from the marrow to the peripheral circulation,967 and conditional targeting of megakaryocyte and platelet integrin α2β1 in mice is associated with reduced MPV.996
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Integrin α5β1 (Also Termed GPIc*/IIa, Fibronectin Receptor, VLA-5 and CD49e/CD29)
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Integrin α5β1 is a receptor that is expressed on a wide variety of different cells and mediates adhesion to fibronectin.804,819,820 It is important for interactions with extracellular matrix, and data from cells other than platelets indicate a role for this receptor in developmental biology and metastasis formation. The RGD sequence in fibronectin is crucial for cell adhesion, but other regions in fibronectin probably also contribute. RGD-containing peptides can inhibit cell adhesion mediated by integrin α5β1. As with other integrin receptors, adhesion depends on the presence of divalent cations. Integrin α5β1 is competent to mediate adhesion of resting platelets to fibronectin,997,998 but its affinity may be modulated by activation.999 The biologic role of this receptor on platelets is not clear. Although it may be involved in hemostasis and/or thrombosis, it is also possible that its function is primarily related to megakaryocyte binding to marrow matrix and proplatelet formation.1000 Integrin α5β1 is not the only fibronectin receptor on platelets, since with appropriate activation, integrin αIIbβ3 can also bind fibronectin.804,1001
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Integrin α6β1 (Also Termed GPIc/IIa, Laminin Receptor, VLA-6 and CD49f/CD29)
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Platelet adhesion to select laminins, which are variably found in basement membranes and extracellular matrix, can be mediated by integrin α6β1.804,1002,1003,1004 Because VWF can bind to some laminins, GPIb can also contribute to platelet adhesion to laminin.1002 This adhesion is best demonstrated with magnesium and manganese; calcium does not support adhesion. This receptor is competent on resting platelets, but its role in platelet physiology is not clear. Mice deficient in integrin α6β1 do not bleed pathologically but are protected against thrombosis.1002 The integrin appears to be able to signal in platelets via PI3 kinase to induce morphological changes.1005 An approximate Mr 67,000 laminin receptor has also been identified on platelets; this receptor is present on other cells as well.1006
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Integrin αvβ3 (Also Termed Vitronectin Receptor and CD51/CD61)
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Integrin αVβ3 receptor shares the same β subunit as integrin αIIbβ3 (GPIIb/IIIa) (see Fig. 112–11).804,855,1007,1008,1009 The integrin αV and αIIb subunits display 36 percent sequence identity.1010 Integrin αVβ3 differs dramatically, however, from integrin αIIbβ3 in its platelet surface density, because there are only approximately 50 to 100 integrin αVβ3 receptors per platelet.1011 The crystal structure of the external domains of integrin αVβ3 alone and in complex with a peptide containing the RGD cell recognition sequence found in a number of ligands have been solved at high resolution.844,845 Such RGD peptides inhibit ligand binding to integrin αVβ3. The most important findings were: (1) the receptor adopts a bent conformation in which the globular headpiece composed of the N-terminal β-propeller region of αV and the βA (I-like) domain of integrin β3, lies near the legs of the integrin αV and β3 subunits, and (2) the RGD peptide binds to the headpiece with the Arg (R) making contact with integrin αV and the Asp (D) making contact with the MIDAS domain in β3. Current evidence suggests that the bent conformation is the inactive one and that activation results in extension of the headpiece and pivoting between the integrin β3 βA and hybrid domains in association with leg separation.827,843,1007,1009 Integrin αVβ3 can mediate adhesion to vitronectin, but only in the presence of magnesium or manganese, not calcium.1011 It can also mediate interactions with fibrinogen, VWF, prothrombin, and thrombospondin.389,1012,1013,1014,1015 Platelet stimulation can activate integrin αVβ3, analogous to activation of integrins αIIbβ3 and α2β1. Activated integrin αVβ3 may uniquely mediate adhesion to osteopontin, a protein found in high concentrations in atherosclerotic plaque.1016 The receptor’s role in platelet physiology is not defined, but it may contribute to the development of platelet coagulant activity.1017
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The integrin αVβ3 receptor is also present on endothelial cells,822,1013 osteoclasts,1018 smooth muscle cells and other cells; it has been implicated in bone resorption,1019,1020,1021 endothelial–matrix interactions,822,1013 lymphoid cell apoptosis,1022 neovascularization,1023 tumor angiogenesis,1023,1024,1025 intimal hyperplasia after vascular injury,1026,1027,1028 sickle cell disease,1029,1030,1031 focal segmental glomerulosclerosis1032,1033 and scleroderma.1034
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The presence or absence of integrin αVβ3 on the platelets of patients with Glanzmann thrombasthenia can help localize the abnormality to either integrin αIIb (if integrin αVβ3 is present in normal or increased amounts) or integrin β3 (if integrin αVβ3 is reduced or absent) (Chap. 121).
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LEUCINE-RICH REPEAT GLYCOPROTEIN RECEPTORS
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GPIb is composed of GPIbα (CD42b) (610 amino acids) disulfide-bonded to two GPIbβ subunits (CD42c) (122 amino acids).801,1035–1043 GPIb appears to exist on the surface of platelets in a 1:1 complex with GPIX (160 amino acids) and a 2:1 complex with GPV (Fig. 112–13). The GPIbα gene is on the short arm of chromosome 17 and the GPIbβ gene is on the long arm of chromosome 22. The GPIX gene is on the long arm of chromosome 3.1044,1045,1046 GPIX is required for efficient surface expression of GPIb,1047 but beyond that, its function is unknown. GPIb/IX is expressed on megakaryocytes and platelets; there is controversy as to whether GPIb/IX is expressed on endothelial cells, either constitutively or after cytokine activation.802 The promoters for GPIb/IX lack TATA or CAAT boxes, but contain binding sites for the GATA and ETS families of transcription factors, which, along with the expression of the cofactor FOG (friend of GATA-1), may account for the limited expression of GPIb/IX.1048–1056
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A genetic polymorphism in GPIbα affects the number of repeating 13-amino-acid units (1, 2, 3, or 4) and produces changes in the molecular weight of GPIbα.1057 The 2 repeat variant is most common, but there is considerable ethnic variation in the frequency of the different numbers of repeats. This molecular weight polymorphism has been linked to the Sib and Ko alloantigens, which have been localized to a T→M variation at amino acid 145 of GPIbα, with M associated with either 3 or 4 repeats and T associated with either 1 or 2 repeats.984 Some, but not all reports suggest an association between the alleles with the larger number of repeats and vascular disease.983,991,1058,1059 Two other GPIbα polymorphisms have been described: (1) C or T at position –5 from the ATG start codon (RS system), and (2) a nucleotide dimorphism at the third bases of the codon for Arg 358.1038,1060,1061 A C at position –5 is present in only 8 to 17 percent of individuals, and more closely resembles the sequence surrounding the ATG start codon (Kozak sequence) considered optimal for translation. In fact, this polymorphism is associated with higher levels of platelet surface GPIb, and may be a risk factor for ischemic vascular disease.1062–1070 GPIb has been implicated as a target antigen in autoimmune thrombocytopenia and in quinine and quinidine-induced thrombocytopenia (Chap. 117).
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GPIbα has a large number of O-linked carbohydrate chains terminating in sialic acid residues,1071 and the latter contribute significantly to the negative charge of the platelet membrane.215 Electron micrographic analysis indicates that GPIb exists as a long flexible rod (approximately 60 nm) with two globular domains of approximately 9 and 16 nm.1072 Thus, GPIb probably extends much further out from the platelet’s surface than does integrin αIIbβ3, which may account for its primacy in platelet adhesion, as well as the increased risk of cardiovascular disease in individuals with longer GPIb molecules because of an increased number of 13-amino-acid repeats. The long extension may also make it susceptible to conformational changes induced by shear forces.801 The extracellular region of GPIbα is readily cleaved by a variety of proteases, including platelet calpains,1073 yielding a soluble fragment named glycocalicin that circulates in normal plasma at 1 to 3 mg/L.1074 In vivo, platelet shedding of glycocalicin from GPIbα is mediated by a disintegrin and metalloprotease (ADAM)-17 (also termed TACE) cleaving a juxtamembrane sequence1075,1076; shedding is controlled by GPIbβ interactions with an unidentified protein, calpain, and reactive oxygen species.1077,1078,1079 Levels of plasma glycocalicin correlate with platelet production and thus can been used to differentiate thrombocytopenia based on decreased platelet production from thrombocytopenia as a result of increased platelet destruction.1080,1081,1082,1083,1084,1085
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GPIbβ and GPIX have free sulfhydryl groups in their cytoplasmic domains that undergo palmitoylation, at least in part, further anchoring the protein to the membrane.1086,1087 The penultimate serine residue at the C-terminus of GPIbα is phosphorylated, providing an attachment site for the signal-complex protein 14–3–3ζ.1088 Similarly, GPIbβ can undergo phosphorylation of Ser 166 in its cytoplasmic domain as a result of protein kinase A activation via cAMP, providing another binding site for 14–3–3ζ (see Fig. 112–13).1089,1090,1091 The cytoplasmic domain of GPIbα connects GPIb to filamin A (actin-binding protein), thus connecting GPIb to the platelet cytoskeleton.993,1092,1093 Coordinated expression of GPIbα and filamin is required for efficient expression of both proteins and imbalances result in abnormalities in platelet size.1094,1095 Alterations in the cytoskeleton can affect GPIb functional activity.1096,1097,1098 14–3–3ζ can bind PI3 kinase and has been implicated in GPIb-mediated intracellular signaling that results in integrin αIIbβ3 activation; Lyn; Vav, Rac1, Alet, and Lim kinase-1 also have been implicated in GPIb/IX–mediated signaling.9,1099,1100,1101 GPIb also appears to be in close proximity to FcγRIIA and the Fc receptor γ-chain, two receptors that can initiate signaling via tyrosine phosphorylation of their cytoplasmic ITAM sequences by Src family kinases and recruitment of the tyrosine kinase syk.1102,1103,1104,1105 Engagement of GPIb by VWF may lead to clustering of GPIb-IX–V complexes in glycolipid-enriched microdomains or lipid rafts, which may serve to concentrate signaling molecules; clustering also increases ligand avidity.1106
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GPIbα has eight leucine-rich repeats in the aminoterminal region of its extracellular domain, whereas GPIbβ and GPIX have one each.1039,1042,1045 These repeats are consensus sequences of 24 amino acids with seven regularly spaced leucines; well-defined disulfide loop sequences flank the repeats.801 Similar leucine-rich repeats are present in a variety of other proteins.
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Crystal structures of the N-terminus of GPIbα (amino acid residue 1–305) alone, and in complex with native and mutated A1 domains of VWF provide important information on the interactions between these proteins (Fig. 112–14).1107,1108 This region of GPIbα adopts a curved shape made up of an N-terminal β-hairpin flanking sequence (finger) containing a C4-C17 disulfide loop (H1-D18), eight leucine-rich repeats (K19-W204), a β-switch region (V227-S241), and a C-terminal sulfated anionic region (D269-D287), with Y276, Y278, and Y279 undergoing posttranslation sulfation.1108,1109,1110 The VWF-A1 domain, which has alternating β strands and α helices organized into a central β-sheet surround by amphiphatic α helices, interacts with the concave face of GPIbα with two areas of tight interactions, at the N-terminal β-hairpin + first leucine-rich repeat (with VWF A1 domain loops α1β2, β3α2, and α3β4), and a more extensive interaction at leucine-rich repeats 5 to 8 + the β switch region (with VWF A1 domain helix α3, loop α3β4, and strand β3). The structure of the VWF A1 domain when not bound to GPIbα differs from that of the bound VWF A1 in that the α1β2 loop protrudes in a way that would prevent interaction with GPIbα.1108 This observation and others related to differences in the ability of different-sized fragments of VWF and GPIbα to interact indicate that other regions of both proteins probably contribute to both the binding and activation of the receptor. The crystal structure of GPIbα with the naturally occurring mutation M239V in the β-hairpin region that results in platelet-type (pseudo-) von Willebrand disease (Chap. 126) has also been obtained,1109 and demonstrates a more stable β-hairpin conformation, which probably accounts for the approximately sixfold increase in binding affinity, primarily through an increase in the association rate. Leucine-rich repeats 3 to 5 do not demonstrate interaction with the normal VWF A domain in the crystal structure, but they are important in ristocetin-induced platelet agglutination, and platelet adhesion at high shear; they do participate to some extent in crystal structures with gain of function mutations in VWF A1.1107,1111,1112 It has been proposed that hydrodynamic forces produced at high shear alter the A1 domain and expose regions that interact with these repeats in GPIb.1113 Other natural and site-directed mutation causing the platelet-type von Willebrand disease pattern of enhanced VWF binding (G233V, V234G, D235V, K237V) also affect the β-hairpin region. A number of Bernard-Soulier syndrome mutations that cause loss of VWF binding to GPIbα localize to the concave face of leucine-rich repeats 5, 6, and 7 (L129P, A156V, and L179del) and to the sides of leucine-rich repeat 2 (C65R and L57P).1110
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The GPIb ectodomain crystal structure has been determined, confirming the four predicted conserved disulfide bonds (C1-C7, C5-C14, C68-C93, and C70-C116), along with the unpaired C122, which crosslinks to GPIbα.1114 The two former disulfides are in the N-capping region and the two latter are in the C-capping region flanking the single leucine-rich repeat.1040 Using a chimeric GPIbβ/GPIX ectodomain protein, the likely contacts between GPIb and GPIX were identified. The structure proposed is a tetramer of one GPIbα, two GPIbβs, and one GPIX in which GPIX interacts with one of the GPIbβ molecules.1037,1040,1043
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Plasma VWF will not bind to GPIb under static conditions unless the antibiotic ristocetin or the snake venom botrocetin is added. The mechanism by which ristocetin induces VWF binding to GPIb is unclear, but effects on VWF as well as on platelet surface charge have been described, and dimerization of ristocetin molecules and multimerization of VWF, as well as stabilization of an A1 domain conformation with high affinity for GPIb have also been implicated.801,1113,1115,1116,1117,1118 Botrocetin binds to VWF, exposing the site that binds to GPIb.1119 Peptide studies implicate the anionic, sulfated tyrosine region of GPIb as the binding site for botrocetin-treated VWF.801
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Unlike integrin αIIbβ3, which requires intact, activated platelets to bind to VWF, GPIb-mediated VWF binding does not require platelet activation or even platelet metabolic integrity, because fixed platelets are readily agglutinated in the presence of VWF and either ristocetin or botrocetin.1116 This observation forms the basis of the assay of plasma VWF activity.
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Platelets will adhere to VWF when the latter is immobilized on a surface, even in the absence of ristocetin or botrocetin.1116,1120,1121,1122 Under these circumstances, the VWF is believed to undergo a conformational change that allows for direct interactions. It may not, however, be necessary to propose a change in VWF conformation as the interaction between VWF and GPIb appears to have both high association and dissociation rates, permitting tethering and translocation on a surface coated with a high density of VWF, but minimal interaction in fluid phase.809 Similarly, VWF associated with fibrin can interact with platelet GPIb without ristocetin or botrocetin.61,1123 The C1C2 domains of VWF appears to contain a fibrin binding site.304
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Shear stress is an important factor in GPIb-mediated adhesion of platelets to immobilized VWF and subendothelial surfaces.1042,1113,1120,1121,1122,1124,1125 Platelets deficient in GPIb or platelets in which GPIb has been blocked with monoclonal antibodies1122,1124 adhere poorly to subendothelial surfaces at all shear rates, but the defect in blood from patients with von Willebrand disease is manifest primarily at higher shear rates.10,11,1122 In what may be a related phenomenon, subjecting platelets to high shear stresses can induce platelet aggregation, which is mediated by VWF binding to GPIb, followed by platelet activation and integrin αIIbβ3-dependent platelet aggregation.13,15,1126 Whether the shear rates generated in vivo in stenotic blood vessels are of sufficient magnitude and duration to produce a similar degree of platelet activation is unknown. It is also uncertain as to whether the effect of shear is acting on GPIb, on VWF, or on both,15,801,809,1042 but shear-induced changes in the structure of VWF, leading to a more extended conformation and conformational changes in the A1 domain, have been defined.1113,1127 GPIb forms catch bonds with VWF, meaning that increasing force first prolongs and then shortens bond lifetimes.1113,1128
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GPIb also functions as a platelet binding site for thrombin.801,1129,1130 The regions between amino acids 216 and 240 and amino acids 269 and 287 were proposed as thrombin binding sites based on biochemical data, with the latter region demonstrating similarity to hirudin, a thrombin-binding protein.801,1131 Sulfation of the three tyrosine residues in the latter region is particularly important for thrombin binding.1093
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Two somewhat different crystal structures of the interactions between thrombin and the negatively charged tail region of GPIb have been reported, but in both cases two molecules of thrombin bind to each GPIb molecule using different regions on thrombin (exosites I and II). This raises the possibility that free thrombin or thrombin adherent to fibrin can cluster GPIb/IX/V complexes.1132
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Binding of thrombin to platelet GPIb appears to contribute to thrombin-induced activation of platelets, even when PAR-1 and PAR-4 are desensitized, and platelets lacking GPIb (Bernard-Soulier syndrome) do, in fact, have blunted responses to thrombin. GPIb has been proposed as the high-affinity binding site for thrombin,1129,1133 but there are only approximately 50 high-affinity thrombin-binding sites and approximately 25,000 GPIb molecules per platelet,1129,1130 raising the possibility that only the subpopulation of GPIb molecules in lipid rafts are able to function in activating platelets.1134 Binding of thrombin to GPIb may also facilitate its effect on one or more of the other thrombin receptors, and there is experimental support for this hypothesis.1135,1136
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GPIb has also been demonstrated to interact with P-selectin in a cation-independent manner.764,1035,1093 Although GPIb shares a number of features with the P-selectin ligand, PSGL-1 (both are sialomucins and have analogous anionic/sulfated tyrosine sequences), the interaction between GPIb and P-selectin appears to be more like the interaction between P-selectin and heparin.1035,1093 In inflamed mesenteric venules in animals, platelets are observed to roll on the activated endothelium1137 and so it is possible that platelet GPIb interacts with endothelial P-selectin in this interaction.1093 PSGL-1, a well-documented ligand for P-selectin on leukocytes, has also been identified on the surface of platelets,1138 and so may also contribute to this interaction.
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GPIbα also binds to high molecular weight kininogen and factor XII, and both of these interactions interfere with thrombin-induced platelet activation.1139,1140 Factor XI also binds to GPIbα, where it undergoes activation by thrombin.1141 Activated leukocyte integrin αMβ2, also can bind to GPIbα via the I-domain of the integrin,1142 and this interaction has been proposed to play an important role in transmigration of leukocytes through platelet thrombi at sites of vascular injury. GPIb plays complex roles in inflammation and endotoxemia in murine models, demonstrating both proinflammatory and antiinflammatory effects.1143,1144 GPIb has also been implicated in supporting metastases in murine models.1145
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GPV, the third member of the GPIb/IX/V complex, has a Mr 82,000 and is composed of 544 amino acids, including 15 leucine-rich repeats. GPV appears to form a noncovalent complex with GPIb, mediated through association of their transmembrane domains,1146 but because the number of GPV molecules on the surface of platelets is approximately 50 percent of the number of GPIb and GPIX molecules,1147 it has been suggested that the basic unit consists of two GPIb molecules, two GPIX molecules, and one GPV molecule.801,1035,1038 GPV is deficient in platelets from patients with Bernard-Soulier syndrome (Chap. 121), but GPV is not required for surface expression of the GPIb/IX complex.1148 A soluble fragment of Mr 69,000 is cleaved from GPV by thrombin, but cleavage does not correlate with thrombin-induced platelet activation.1149 Platelets from mice lacking GPV appear to respond more actively to thrombin and ADP than wild-type mice, raising the possibility that GPV inhibits platelet activation.1150 The platelets from these mice also adhere to immobilized VWF and can bind VWF in the presence of botrocetin, indicating that GPV is not required for the interaction between VWF and the GPIb/IX/V complex.1150 It has been proposed that removing a portion of GPV by thrombin proteolysis allows thrombin access to GPIbα, thus facilitating its ability to activate platelets. In support of this model, thrombin’s ability to activate platelets does not require proteolytic activity if GPV is absent, suggesting a direct nonproteolytic effect mediated via GPIbα.1151
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IMMUNOGLOBULIN FAMILY OF CELL-SURFACE ADHESION RECEPTORS AND THEIR ASSOCIATED MEMBRANE PROTEINS
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Platelet-Endothelial Cell Adhesion Molecule-1 (Also Termed CD31)
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PECAM-1 is a transmembrane glycoprotein of the immunoglobulin gene family with six immunoglobulin-like domains of the C2 group and a Mr 130,000.1152,1153,1154,1155 In addition to platelets and endothelial cells, PECAM-1 is expressed on monocytes, myeloid cells, and some lymphocyte subsets. There are approximately 8000 PECAM-1 molecules on the surface of platelets.1156 PECAM promotes homophilic interactions via a homophilic binding domain in the immunoglobin-like repeats. The cytoplasmic tail of PECAM is 118 amino acids in length and contains a palmitoylation site (C595), an immunoreceptor tyrosine-based inhibitory motif (ITIM) including Y663, an immunoreceptor tyrosine-based switch motif (ITSM) including Y686, and a lipid-interacting α helix that contains Y686 and S702, which undergoes inducible phosphorylation.1152,1157 Upon phosphorylation, the ITIMs recruit and activate phosphatases, such as SHP-2 and to a lesser extent SHP-1, SHIP, and PP2A,1158 via their SH2 domains.1152 PECAM-1 undergoes homotypic interactions that lead to signaling and crosslinking.1159 PECAM-1 activation overall thus induces inhibitory activity as the phosphatases counteract the effects of stimulating kinases, but are complex and agonist specific. PECAM-1 activation decreases platelet responses to ADP and thrombin and PECAM-1 platelet expression correlates inversely with platelet sensitivity to these agonists.1160 PECAM-1 also negatively regulates collagen-induced platelet activation mediated by the ITAM-bearing GPVI/FcRγ-chain complex, GPIb/IX/V signaling, and laminin-induced activation.1159 Platelets from mice lacking PECAM-1 are hyperresponsive to subthreshold doses of collagen, and when compared to wild-type mice, form larger platelet thrombi on VWF and in experimental settings in vivo.
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Crosslinking PECAM-1 molecules on the platelet surface with antibodies enhances platelet adhesion and aggregate formation, suggesting that under certain circumstances PECAM-1 might be a costimulatory agonist, working in concert with platelet integrin αIIbβ3.1161 Moreover, mice lacking PECAM-1 can undergo normal inside-out activation of integrin αIIbβ3, but have a partial defect in integrin αIIbβ3-mediated outside-in signaling.1162 PECAM-1 crosslinking may also lead to GPIb internalization, resulting in decreased platelet adhesion.1163
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In endothelial cells, PECAM-1 is localized to the contact areas between endothelial cells, in the lateral border recycling compartment, where it is involved in controlling stimulus-specific transmigration of leukocytes.1155,1164 It appears to be capable of both homotypic and heterotypic interactions, with the latter mediated by CD177 on neutrophils (and perhaps glycosaminoglycans, integrin αVβ3, or CD38) interacting with the fifth or sixth PECAM-1 immunoglobulin domain.1155,1165 PECAM-1 engagement triggers signaling and leukocyte integrin receptor activation that facilitates transmigration, with activation of the laminin receptor, integrin α6β1, of particular importance. Endothelial PECAM-1 is also important in maintaining vascular integrity and endothelial and leukocyte PECAM-1 mediate both proinflammatory and antiinflammatory phenomena in model systems.1155
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Triggering Receptors Expressed on Myeloid Cells–Like Transcript-1
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Triggering receptors expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) is a receptor whose external domain is homologous to those in the family termed TREM. Like those receptors, it contains a single V-set immunoglobulin domain, but its cytoplasmic domain is much longer, is palmitoylated and carries a canonical ITIM capable of becoming phosphorylated and binding the Src homology-containing protein, tyrosine phosphate-1 (SHP-1).627,1166 The phosphatase can then dephosphorylate signaling molecules, leading to inhibition of platelet activation. PECAM-1 has a similar ability to bind SHP-1. TLT appears to be restricted in expression to platelets and megakaryocytes. It is primarily in α-granule membranes in resting platelets and joins the plasma membrane when platelets are activated.
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GPVI is a Mr 62,000 transmembrane glycoprotein of 316 amino acids.18,804,1167,1168 It belongs to the immunoglobulin superfamily, and is the major platelet signaling receptor for collagen. It may also mediate platelet interactions with monocytes by binding the ligand EMMPRIN.1169 GPVI on the platelet surface exists in a complex with Fc receptor (FcR) γ-chain. Because the latter is a dimer, two GPVI molecules associate with one FcR γ-chain, forming a high-affinity complex.1168 The GPVI extracellular region contains two immunoglobulin C2-like domains and its transmembrane domain contains an Arg residue that is essential for association with the FcRγ-chain. The 51-amino-acid cytoplasmic domain contains a proline-rich sequence that binds SH3 (Src homology 3) domains of Src family tyrosine kinases. GPVI signals through the FcRγ-chain, which contains an ITAM. An unpaired thiol in the cytoplasmic tail of GPVI can undergo oxidation, resulting in homodimer formation,1170 required for high-affinity interactions with collagen peptides and GPVI-mediated signaling.1171,1172 Resting platelets have approximately 29 percent of their GPVI molecules in dimers and interactions with CRPs or thrombin activation increase the percentage of GPVI in dimers.1171 When GPVI binds collagen, the ITAM domain of the FcRγ-chain becomes phosphorylated by the Src kinases Fyn and/or Lyn, resulting in the formation of large complex of signal-transducing proteins (for a discussion of the role of GPVI as a receptor for collagen, see “Signaling Pathways in Platelets” below.).1041,1104 GPVI is required for stable platelet thrombus formation on collagen surfaces in vitro. Mice lacking GPVI have a relatively mild phenotype and are protected from thrombosis in some but not all experimental models. GPVI and FcRγ-chain appear to play important roles in ferric chloride-mediated arterial thrombosis in mice, but not in laser-induced thrombosis, perhaps because the former, but not the latter, injury elicits collagen exposure along the damaged vessel. Inherited and acquired defects in human platelet GPVI have been reported (Chap. 121) and the associated bleeding disorders have been variably described as mild to severe.1172,1173,1174,1175,1176 Two alternatively spliced forms and several polymorphisms have been identified for GPVI and variably linked to alterations in platelet function or risk of thrombotic disease.1177,1178
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The FcRγ-chain1179 exists as a homodimer of Mr 20,000 that physically and functionally associates with GPVI1180 and GPIb/IX.1102 In mouse platelets, the absence of FcRγ-chain results in lack of surface expression of GPVI. The FcRγ-chain, along with FcγRIIA, are the only known platelet proteins with ITAMs.1104 Phosphorylation of the ITAM domain serves to recruit proteins with Src homology 2 (SH2) domains, which are essential for collagen-mediated signaling through the GPVI/FcRγ-chain pathway.1041,1104,1181 The FcRγ-chain may also contribute to GPIb/IX-mediated intracellular signaling after VWF binding.1035,1102,1105
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Fcγ Receptor IIA (FcγRIIA, Also Termed CD32)
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The FcγRIIA is a low affinity immunoglobulin receptor of Mr 40,000 that is widely distributed on hematopoietic cells.804 Three different mRNA transcripts (A, B, and C) make similar FcγRIIA molecules1182 and these are preferentially expressed on different cells. FcγRIIA contains an ITAM domain and thus may be important for signaling by its associated proteins, including GPIb and select integrins, as well as through direct stimulation by immune complexes. Crosslinking of FcγRIIA initiates tyrosine phosphorylation, PI metabolism, activation of PLCγ2, calcium signaling, and cytoskeletal rearrangements.960,961 FcγRIIA appears to be in close proximity to the GPIb/IX/V complex in lipid rafts,212 and signal transduction that accompanies VWF binding to GPIb may be mediated at least in part through FcγRIIA.885,971 FcγRIIA is also be important in mediating integrin αIIbβ3 outside-in signaling, including effects on platelet spreading, clot retraction, and thrombus formation.1183,1184 Platelet 12(S)-lipoxygenase (LOX) is required for platelet activation mediated by FcγRIIA.1185
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The FcγRIIA on platelets may bind immune complexes generated in certain diseases, and by engaging these complexes the platelets may become sensitized to other stimuli.1186,1187,1188 It may also provide a second binding site for antibodies that bind to platelets via their antibody-binding site (see “CD9” below). This second interaction can potentially lead to bridging between platelets, with the antibody binding to an antigen on one platelet and an FcγRIIA receptor on another platelet.1189 It is also possible that antibodies can bind to both an antigen and an FcγRIIA on a single platelet. These interactions can lead to platelet activation through engagement of FcγRIIA, followed by crosslinking of FcγRIIA receptors, which can lead to tyrosine phosphorylation, PI metabolism, activation of PLCγ2, calcium signaling, and cytoskeletal rearrangements.1190,1191 This type of interaction appears to play an important role in heparin-induced thrombocytopenia (Chap. 117). FcγRIIA undergoes proteolysis when platelets are activated and FcγRIIA proteolysis has been proposed as an assay for heparin-induced thrombocytopenia.1192,1193 Cooperation between FcγRIIA and C1q receptor has been reported.1194 A variety of viruses and bacteria can interact with, and activate platelets and this is variably mediated by FcγRIIA, with or without immunoglobulin.795,1195,1196 FcγRIIA may also contribute to cancer cell activation in platelets.1197
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FcγRIIA expression on platelets shows considerable variation among individuals (approximately 600 to 1500 molecules per platelet), and this variation correlates with FcγRIIA-mediated function.1188 This variation in receptor density may explain individual differences in immune-mediated disorders such as heparin-induced thrombocytopenia with thrombosis.1198 An H131R polymorphism within FcγRIIA affects the binding of different IgG subclasses.1199,1200 The H131R polymorphism may also have clinical significance because the R131 allele is associated with increased binding of activation-dependent antibodies to platelets.1201 A variety of associations have been identified between the H131ER polymorphism and different aspects of heparin-induced thrombocytopenia and immune thrombocytopenia, but the data differ from study to study and no consensus has yet emerged.1202,1203,1204,1205,1206,1207
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Intercellular Adhesion Molecule-2 (CD102)
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ICAM-2, a member of the immunoglobulin family of receptors, is an endothelial cell ligand for the β2-integrin αLβ2 (LFA-1) on lymphocytes and myeloid cells.1208 Approximately 2600 ICAM-2 molecules are present on platelets, distributed on the membrane surface and open canalicular system.1208 Platelet ICAM-2 may contribute to platelet-leukocyte interactions (see “Platelet–Leukocyte Interactions” below).
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Platelets express the high affinity IgE receptor FcεRI and appear to participate in both defense against parasitic diseases, including malaria, and allergic phenomena.799,1209,1210,1211
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Junctional Adhesion Molecule-A (A Also Termed F11)
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JAM-A was identified on platelets by the ability of a monoclonal antibody directed against the receptor to initiate platelet activation via crosslinking to FcγRIIA.1212,1213,1214,1215,1216 It is phosphorylated during platelet activation and loss of JAM-A in a mouse model results in a prothrombotic phenotype.1217 JAM-A appears to inhibit outside-in signaling via integrin αIIbβ3 by recruiting Csk, which, in turn, phosphorylates Src at Y529.1217,1218 It is also able to interact with the integrin αLβ1 receptor on leukocytes, and in endothelial cells it participates in tight junction formation and leukocyte recruitment and transmigration.920
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Junctional Adhesion Molecule-C
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The JAM-C transmembrane protein has an Mr of 43,000 and 279 amino acids. It contains two C2-type immunoglobulin domains in its extracellular domain and three potential tyrosine phosphorylation sites in its cytoplasmic domain.767,920 JAM-C is expressed on platelets but not granulocytes, monocytes, lymphocytes, or erythrocytes. It shares 32 percent homology with JAM-A. Based on monoclonal antibody binding studies, platelets contain approximately 1600 copies of JAM-C. Platelet JAM-C acts as a counterreceptor for leukocyte integrins αMβ2 and αXβ2 and contributes to platelet–leukocyte interactions under some conditions.767 Its precise role in platelet physiology is uncertain, but it has been implicated in binding CD34 stem cells.1219
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LECTIN-CONTAINING RECEPTORS
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P-Selectin (Also Termed GMP140, PADGEM, and CD62P)
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P-selectin, which has a Mr of 140,000, is a glycoprotein present in α-granule membranes in resting platelets that joins the plasma membrane when platelets are activated.759,1220,1221,1222 Approximately 13,000 P-selectin molecules are detected by antibodies on the surface of activated platelets. The expression of P-selectin on circulating platelets has, therefore, been used as an indicator of their in vivo activation.1223,1224 It is also present in the Weibel-Palade body membranes of endothelial cells; as in platelets, it joins the plasma membrane when endothelial cells are activated.759,1222
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P-selectin has a modular structure in which the aminoterminal region has a calcium-dependent lectin domain that binds carbohydrates. Adjacent to the lectin domain is an EGF domain, followed by nine repeats that are homologous to complement regulatory proteins (“sushi” domains), a transmembrane domain, and a cytoplasmic domain.759,1220 The cytoplasmic domain contains Ser, Thr, Tyr, and His residues that can be phosphorylated. In addition, a Cys residue becomes acylated with stearic or palmitic acid. Alternatively spliced forms of P-selectin may be produced in which sushi domains are omitted. The selectin family also includes E-selectin (ELAM-1; CD62E), which is expressed on the surface of activated endothelial cells, and L-selectin (LAM-1; CD62L), which is expressed on the surface of myeloid and lymphoid cells.1225
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Soluble P-selectin is present in plasma from humans and mice. Alternative splicing generates a soluble form of human P-selectin that lacks the transmembrane domain.1226 In mice, at least a portion of soluble P-selectin is derived from proteolytic cleavage of surface P-selectin by an unidentified protease.1227
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Recognition of ligand by P-selectin requires specific carbohydrate and protein structures. Fucose and sialic acid are important carbohydrate components, with sialyl-3-fucosyl-N-acetyllactosamine (SLex; CD15S) a preferred ligand structure.756,1228,1229,1230 Myeloid and tumor cell sulfatides may also act as ligands for P-selectin.1231,1232 PSGL-1, a mucin-like transmembrane glycoprotein homodimer (Mr 220,000) expressed on neutrophils, monocytes, lymphocytes, and to a small extent on platelets, is an important ligand for P-selectin.1138,1233,1234,1235 Both sulfation of tyrosine residues contained in an anionic region and branched fucosylation of O-linked carbohydrates are required for optimal binding to P-selectin.
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P-selectin can mediate the attachment of neutrophils and monocytes to platelets and endothelial cells. Thus, neutrophils and monocytes may be recruited to sites of vascular injury where platelets deposit and become activated (see “Platelet–Leukocyte Interactions” below). Platelet P-selectin can also recruit procoagulant monocyte-derived microparticles containing both PSGL-1 and tissue factor to growing thrombi in vivo.1236 Binding of P-selectin to PSGL-1 on monocytes can trigger tissue factor synthesis1237 and infusing a P-selectin chimeric molecule into mice results in the generation of procoagulant microparticles.781 In a reciprocal fashion, P-selectin engagement of PSGL-1 may lead to platelet activation.1238 Soluble P-selectin may also promote a prothrombotic state in humans by increasing tissue factor–expressing microparticles in plasma. Indeed, the risk of future cardiovascular events is elevated in apparently healthy women with the highest levels of soluble P-selectin.1239
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In intact blood vessels, the rapid on and off rates of the interactions between PSGL-1 on neutrophils and P-selectin on endothelial cells allows leukocytes to roll on the endothelium, the first step in leukocyte transmigration (Chap. 66).1240 The rapid upregulation of P-selectin after endothelial cell activation allows for a quick response. Platelets have been reported to roll on activated endothelium, and this appears to result from an interaction between endothelial P-selectin and perhaps either platelet GPIbα1093,1137 or platelet PSGL-1.1138,1241 Upon their corelease from endothelial Weibel-Palade bodies, P-selectin may tether ultralarge VWF to the surface of activated endothelium, and thereby promote platelet GPIbα-mediated platelet rolling.1242
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Genetic and pharmacologic targeting of P-selectin or PSGL-1 in experimental animal models suggests that these receptors may modulate thrombolysis, sickle cell vasoocclusion, restenosis, deep venous thrombosis, cerebral ischemia and infarction, atherosclerosis, metastasis, and thrombotic glomerulonephritis (reviewed in Refs. 1243,1244,1245,1246).
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C-Type Lectin-Like Receptor-2
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Podoplanin is a sialoglycoprotein present on a variety of tumor cells, lymphatic endothelial cells, kidney podocytes, lung epithelial cells, lymph node stromal cells, and the choroid plexus epithelium that can aggregate platelets.1247,1248,1249,1250 Its receptor on platelets is CLEC-2, a C-type lectin-like receptor selectively expressed on megakaryocytes and platelets (approximately 2,000 copies per platelet) that binds podoplanin and the snake venom platelet-activating protein rhodocytin.1251,1252,1253 The cytoplasmic tail of CLEC-2 contains an atypical ITAM (hemITAM) with a single YITL sequence that can be tyrosine phosphorylated by Src kinases when platelets are activated. Because CLEC-2 exists as a dimer, it can supply two ITAMS and lead to activation of Syk and, ultimately, PLCγ2.1254 This signaling system is similar to that of GPVI in combination with the FcRγ-chain. Activation of CLEC-2 leads to proteolytic cleavage of GPVI and FcγRIIa.1255 In experimental tumor models, inhibiting the podoplanin/CLEC-2 system reduces metastases.1256 CLEC-2 interaction with podoplanin on lymphatic endothelial cells, followed by platelet activation and CLEC-2-podoplanin clustering, is required for the separation of blood and lymph vessels during development.1257,1258,1259 CLEC-2 also plays a role in lymph node development and maintenance.1260 HIV-1 can also bind to CLEC-2.
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Tetraspanins are a family of four-transmembrane-domain-containing proteins that have conserved Cys residues that form crucial disulfide bonds. The extracellular and intracellular loops in these proteins contain many motifs that mediate interactions with other proteins.1261 While the specific function(s) of tetraspanins is not yet clear, these proteins are able to associate with several membrane proteins and have been reported to modulate integrin function, perhaps in part by organizing membrane and intercellular signaling molecules in cholesterol-associated microdomains distinct from lipid rafts.1262 CD9, CD63, and CD151 have juxtamembrane Cys residues that can be palmitoylated and this modification may contribute to assembly of complexes with other proteins and localization to lipid microdomains.1263 Studies in mice implicate CD151 and TSSC6 in outside-in signaling of integrin αIIbβ3.1264 Oligomers of tetraspanins are known to facilitate the formation of larger complexes of membrane proteins that could serve as scaffolds for several platelet signaling events.1265 CD9 is the most abundant platelet tetraspanin (approximately 40,000 molecules per platelet), followed by CD151, Tspan9, and CD63.1266 The levels of TSSC6 are not known.
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CD9 (5H9; BA2; P24; GIG2; MIC3; MRP-1; BTCC-1; DRAP-27; TSPAN29)
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CD9 is a 228-amino-acid tetraspanin that is present on platelets, endothelial cells, smooth muscle cells, cultured fibroblasts, some lymphoblasts, eosinophils, basophils, and other cells.1267,1268,1269 It colocalizes with integrin αIIbβ3 on the inner surface of α granules in resting platelets and on pseudopods of activated platelets.1270 Binding of monoclonal antibodies specific for CD9 to platelets results in platelet aggregation by triggering phosphatidylinositol metabolism via a mechanism that also requires binding to the platelet FcγRIIA receptor.1271,1272,1273 The platelet activation induced by the binding of such antibodies requires external calcium and results in an association between CD9 and integrin αIIbβ3.1274 CD9 has been proposed to play a role in microparticle release from platelets.1275 Studies in mice lacking CD9 suggest that CD9 is a negative regulator of integrin αIIbβ3 signaling, as the mice have enhanced platelet aggregation, fibrinogen binding, and thrombus formation.1276
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CD63 (Also Termed Granulophysin and LAMP-3)
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CD63 (Mr 53,000) appears to be present in both lysosomal and dense granule membranes in platelets.310,1263,1277 CD63 is also present in Weibel-Palade bodies in endothelial cells, the lysosomal membranes of a variety of other cells, as well as the membranes of melanosomes. It appears on the surface membrane when platelets are activated, making it a useful marker for platelet activation.310,1224 CD63 colocalizes with integrin αIIbβ3 and CD9 on the surface of activated platelets in a process that appears to require CD63 palmitoylation.1263 CD63 is markedly reduced or absent from the dense bodies of patients with Hermansky-Pudlak syndrome,1277 who have oculocutaneous albinism and a defect in platelet dense bodies (Chap. 121). The amino acid sequence of CD63 has been deduced from complementary DNA (cDNA) cloning.1278
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CD151 (Also Termed GP27, MER2, RAPH, SFA1, PETA-3, and TSPAN24)
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CD151, a glycoprotein of Mr 27,000 is present on platelets, endothelial cells, and many other cells.1279,1280,1281 Antibodies to CD151, like those to CD9, can initiate platelet aggregation by binding to both CD151 and FcγRIIA.1280 The role of CD151in platelet physiology remains to be firmly established but it may participate with FcγRIIA as a signal transduction complex.1280 CD151 appears to functionally associate with integrin αIIbβ3 and, in mice, loss of CD151 impairs platelet aggregation, clot retraction,1282 and thrombus formation.1283
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TSSC6 (PHMX, PHEMX FLJ17158, FLJ97586, MGC22455, TSPAN32)
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TSSC6 is a 340-amino-acid tetraspanin that is expressed in marrow, spleen, thymus, and several hematopoietic cell types.898 It is present in platelets and has been reported to interact with integrin αIIbβ3. Mice deficient in TSSC6 display a slightly prolonged bleeding time and a significantly increased rebleeding.1265 Platelets lacking TSSC6 show impaired aggregation and clot retraction.
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GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS (CD55, CD59, CD109, PRION PROTEIN)
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At least five separate platelet proteins are attached to the membrane through a GPI link. These include proteins involved in complement regulation (CD55, decay accelerating factor, and CD59, membrane inhibitor of reactive lysis)1284; CD109, a Mr 170,000 protein present on platelets, endothelial cells, hematopoietic cells, and fibroblasts that carries both ABO oligosaccharides and an alloantigen (HPA-15, Gov) involved in neonatal isoimmune thrombocytopenia1285,1286; and a Mr 500,000 protein of unknown identity. Patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormalities in the GPI anchor and thus variably lack all of the GPI-linked proteins. The diagnosis of PNH can be established by assessing platelet expression of these proteins.897,1287,1288 Patients with PNH have been reported to have platelet function abnormalities,1287 raising the possibility that one or more of these proteins has a role in platelet function, but no specific platelet function roles have yet been assigned to the proteins. Of particular interest is the presence of the normal prion protein, which is a Mr 27,000 to 30,000 GPI-linked protein that is both upregulated and shed from the platelet surface with platelet activation.1289,1290,1291,1292 In fact, platelets contain the majority of the prion protein present in normal blood.
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TYROSINE KINASE RECEPTORS
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Eph Kinases and Ephrin Ligands
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Eph kinase receptors comprise the largest family of cell surface-associated tyrosine kinases with 14 members identified in mammals. Eph kinases have a conserved structure consisting of an N-terminal extracellular ephrin-binding domain, two fibronectin type II repeats, and intracellular kinase, sterile α motif (SAM), and PDZ binding domains [defined by the first three proteins to display this protein–protein domain, post synaptic density protein (PSD95), Drosophila disk large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1)]. A total of eight ephrins have been identified that serve as cell-surface ligands for the Eph kinases. In general, Eph A kinases recognize ephrins that contain a GPI anchor (ephrin A family), while Eph B kinases bind to ligands with a transmembrane domain (ephrin B family). The Eph receptors and the ephrins appear to signal bidirectionally at sites of cell-to-cell contact. Platelets contain Eph kinases EphA4 and EphB1, and their ligand ephrin B1, as well as EphB2.276,1293 Messenger RNA for ephrinA3 has also been detected in platelets, but confirmation of the presence of ephrinA3 protein in platelets is lacking. Forced clustering of either Eph kinases or ephrins in platelets promotes cytoskeletal reorganization, adhesion, granule secretion, and Rap1b activation in concert with other platelet stimuli.1293,1294 Eph kinase–ephrin interactions may stabilize platelet aggregates and thrombus formation after platelet–platelet contact has occurred.276,1295
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Thrombopoietin Receptor (c-mpl, CD110)
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The thrombopoietin receptor (c-mpl; Mr 80 to 85,000) is expressed at low levels on platelets (approximately 25 to 224 per platelet) and binds thrombopoietin with high affinity. (KD approximately 0.50 nM).1296,1297,1298,1299 Steady-state plasma levels of thrombopoietin are maintained, in part, by platelets and megakaryocytes, which bind thrombopoietin via the thrombopoietin receptor and then internalize and degrade the growth factor. Additional mechanisms for regulation of thrombopoietin levels have been described (Chap. 111). Although its major function is to stimulate megakaryocyte growth and maturation (Chap. 111), thrombopoietin also is able to sensitize platelets to activation by agonists.1300,1301,1302,1303,1304,1305 Mutations of the receptor have been associated with inherited thrombocytopenia (Chap. 117) and myeloproliferative neoplasms (Chaps. 83 to 85).1306,1307 It can also contribute to hematopoiesis through effects on hematopoietic stem cells and other progenitors.
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CD36 (GPIV) is a Mr 88,000 glycoprotein that is highly, but variably, expressed on platelets (approximately 20,000 copies per platelet).1308,1309,1310,1311,1312,1313 The nucleotide sequence of CD36 (GPIV) cDNA encodes a protein of 471 residues with a predicted Mr of 53,000 and 10 potential N-linked glycosylation sites,1314 accounting for the difference between predicted and experimentally determined Mr. It is unusual in having two putative transmembrane domains and two short cytoplasmic tails. The cytoplasmic regions may associate with intracellular tyrosine kinases of the Src family and undergo phosphorylation.1315 Antibodies to CD36 (GPIV) have been reported to produce neonatal alloimmune thrombocytopenia (Chap. 117).1316 Biochemical data suggest that it may form dimers and multimers.1317 Increased platelet surface expression of CD36 (GPIV) has been described in patients with myeloproliferative neoplasms.1318 CD36 (GPIV) is also expressed on phagocytic cells (with the exception of neutrophils), fat and muscle cells, cardiac myocytes, and microvascular endothelial cells. The phosphorylation status of the extracellular region of the protein may control its ligand-binding properties,1319 offering a potential explanation for some of the variable results obtained under different conditions.1308,1319,1320
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CD36 (GPIV) plays an important role in long-chain fatty acid transport in the heart, fat, and muscle, and may contribute to atherosclerosis and insulin sensitivity.1321,1322 Oxidized low-density lipoproteins (LDL), which can be produced by the effects of endothelial cell or platelet nitric oxide (NO) on LDL, bind to CD36 and, perhaps in concert with scavenger receptor (SR)-A, can increase platelet reactivity to agonists via signal transduction mediated in part by Src kinases and a MAPK.1323,1324,1325 The variability in platelet CD36 expression may account for the variability in platelet hyperreactivity in response to elevated levels of oxidized LDL.1326 CD36 can also mediate microparticle binding to platelets, which augments platelet-mediated thrombosis in model systems.1327 Thus, CD36 has been reported to contribute to atherogenesis, diabetes, the metabolic syndrome, angiogenesis, and inflammation.1328,1329,1330,1331 CD36 also interacts with the S100 calcium-modulated protein family member myeloid-related protein (MRP)-14 (also known as S100A9), which can be released from activated neutrophils and platelets. It has been proposed as a platelet receptor for thrombospondin1332 and collagen,1333,1334 but the functional significance of these interactions remains unclear because individuals who lack CD36 on an inherited basis (Naka-negative) do not have a bleeding disorder1335 (Chap. 121). CD36 may play a role in the thrombospondin-mediated interaction reported between platelets and sickle erythrocytes,1336 apoptosis, innate immunity, and in the binding of Plasmodium falciparum-infected erythrocytes to endothelial cells and monocytes.1310,1314
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Scavenger Receptor-BI (SCARB1; CLA-I)
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The class B SR-BI (CLA-I) is related to CD36 and is expressed on platelets, endothelial cells, and hepatocytes.1313 It transports the cholesteryl esters from high-density lipoprotein (HDL) cholesterol and facilitates bidirectional flux of free cholesterol between cells and lipoproteins. Oxidized, but not unoxidized, HDL can inhibit platelet aggregation via binding to SR-BI.1337 SR-BI has many other lipid ligands, however, and it is uncertain how these interact under physiologic conditions. A number of mutations are associated with elevated HDL levels.1338 A heterozygous missense mutation has been associated with increased platelet unesterified cholesterol and both increased and decreased platelet function.1338 Mouse studies indicate that disrupting the SR in nonhematopoietic tissues can affect platelet function via alterations in plasma lipids and alterations in the platelet SR can protect against hyperactivity induced by increased platelet cholesterol content.1326
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CD40 Ligand (CD40L, CD154) and CD40
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CD40 ligand (CD40L, CD154) is a trimeric transmembrane protein (Mr 33,000) of the tumor-necrosis family that localizes to α granules in resting platelets and rapidly appears on the surface of platelets upon activation. Within minutes to hours of platelet activation, an Mr 18,000 fragment of CD40L is released from the platelet surface, perhaps mediated in part by matrix metalloproteinase (MMP-2) bound to integrin αIIbβ3.1339 This soluble form of CD40L circulates as a trimer. The bulk of soluble CD40L in plasma is derived from activated platelets and, hence, can serve as a marker for platelet activation in vivo. Elevated levels of soluble CD40L are observed in acute coronary syndromes, following percutaneous coronary intervention, in the setting of coronary artery bypass surgery, and in peripheral vascular disease1340 (reviewed in Refs. 1341 and 1342). Soluble CD40L activates neutrophil integrin αMβ2, enhances neutrophil adhesion, and induces the neutrophil oxidative burst.1343 Moreover, elevated levels of soluble CD40L are associated with recurrent cardiovascular events in the setting of acute coronary syndromes1340,1344 and restenosis following percutaneous coronary intervention.1345 CD40L and, to a lesser extent, its counterreceptor CD40 have been implicated in the progression of atherosclerosis in animal models.1346,1347
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The extracellular portion of CD40L binds to CD40, a Mr 48,000 transmembrane receptor. Approximately 600 to 1000 copies of CD40 are present on both resting and activated platelets,1348 and while CD40L has been reported to initiate platelet activation via binding to CD40,1349 the functional significance of CD40–CD40L interactions in platelet physiology remains to be determined. CD40L also contains a KGD sequence (RGD in mice) that has been implicated in binding to integrin αIIbβ3. In mice, CD40L–αIIbβ3 interactions appear to stabilize thrombus growth,1348 perhaps by activating receptor mediated signaling.1350 Additionally, integrin αIIbβ3 antagonists block the release of soluble CD40L from activated platelets. Both platelet-associated and soluble CD40L may stimulate leukocytes to release proinflammatory cytokines; CD40L may also inhibit endothelial cell migration after vascular injury.1351 The inhibitory effects of CD40L on reendothelialization may partially explain why elevated levels of soluble CD40L are associated with higher rates of clinical restenosis.1345 Finally, platelet CD40L may modulate adaptive immunity by serving as a costimulatory signal for antigen presenting cells.1352,1353
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Fas Ligand, LIGHT and TRAIL
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Fas ligand (FasL), LIGHT (also termed TNF superfamily member 14), and TNF-related apoptosis-inducing ligand (TRAIL), along with CD40L, belong to the TNF family of cytokines.1354 With activation, platelets express FasL, LIGHT, and TRAIL on their surface and release soluble forms of these receptors,1354,1355,1356 analogous to activation-dependent CD40L platelet expression and release. The receptor Fas (Apo-1, CD95), is expressed on a wide variety of normal and malignant cells. Engagement of Fas by FasL initiates signaling that results in apoptosis, and this process is important in embryonic development, cellular hemostasis, and immune regulation.1354 The surface-expressed FasL on platelets is biologically active and can initiate apoptosis. The soluble form of FasL may act as an inhibitor of apoptosis induced by surface-expressed FasL.1354 Similarly, platelet-derived LIGHT is biologically active and can initiate inflammatory responses in monocytes and endothelial cells.1356
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Lysosome-Associated Membrane Proteins 1 and 2 (CD107a, CD107b)
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LAMP-1 and LAMP-2 are lysosome-associated membrane proteins that are approximately 30 percent homologous and constitute approximately 50 percent of lysosomal membrane proteins.1357 They are integral membrane glycoproteins of Mr 110,000 and 120,000, respectively, that are contained within lysosomal membranes.1358 When platelets undergo the release reaction, they join the plasma membrane. Each protein has two extracellular disulfide-bonded loops containing 36 to 38 amino acids. The loops are separated by a region rich in Pro and Ser that shares homology with the hinge region of IgA. There are multiple N-linked glycosylation sites on each glycoprotein and they contain more than 60 percent carbohydrate. Among the carbohydrate residues are polylactosaminoglycans that may possess sialylated Lewisx structures, which are thought to interact with selectins. LAMP-1 and LAMP-2 play roles in control of lysosome fusion in autophagosomes and phagosomes.1357
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Platelets have several receptors for C1q, a Mr 460,000 glycoprotein composed of six globular domains attached to a short collagen-like triple helix.1359,1360,1361 One is for the collagen-like domain (cC1qR, Mr 60,000 to 67,000 nonreduced and 72,000 to 75,000 reduced), and another is for the globular domain (gC1qR, Mr 28,000 to 33,000).1362,1363 A third receptor of Mr 126,000 enhances phagocytosis.1364 C1q circulates with C1r and C1s as a calcium-dependent complex, but interaction with immune complexes leads ultimately to dissociation of the complex and release of free C1q, with its collagen-like domain exposed. cC1qR has sequence homology to calreticulin and can modulate platelet-collagen interactions at low collagen concentrations. It may also localize immune complexes, and when crosslinked by aggregated C1q, it can initiate platelet activation, aggregation, secretion, and expression of platelet coagulant activity.1365,1366 Thus, the binding of C1q monomers to platelets inhibits collagen-induced platelet aggregation but has little effect on platelet adhesion to collagen.1367 C1q multimers support platelet adhesion and can induce aggregation via activation of integrin αIIbβ3.1368 C1q can also augment platelet aggregation induced by aggregated IgG.1194 The gC1qR may self-associate to form a doughnut-shaped ternary complex.1369 In addition to binding C1q, this receptor can bind S. aureus protein A on endothelial cells, where it functions as a receptor for high-molecular-weight kininogen.1363 It may, therefore, participate in contact activation.
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GMP-33 (Thrombospondin N-Terminal Fragment)
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A Mr 33,000 α-granule membrane protein was initially identified as an activation-dependent protein that joins the plasma membrane when platelets undergo the release reaction. Approximately 4000 antibody molecules directed against GMP-33 bind to resting platelets, and 19,000 bind to activated platelets.1370 Subsequent studies identified this antigen as a membrane-associated fragment from the N-terminal of thrombospondin.1371
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Leukosialin, Sialophorin (CD43)
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Leukosialin, a glycoprotein of Mr 90,000, may act as a ligand for ICAM-1.1372 It is expressed on myeloid and some lymphoid cells. Abnormalities in leukosialin have been described in Wiskott-Aldrich syndrome (Chap. 121).
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Toll-Like Receptors 1, 2, 4, 6, 9
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Toll-like receptors (TLRs) are involved in innate immunity by virtue of their ability to sense products of protozoa, fungi, viruses, and bacteria, including endotoxin (lipopolysaccharide [LPS]), and then activate intracellular signaling pathways to initiate the inflammatory response.1373 TLRs 1, 2, 4, 6, and 9 have been identified in platelets.1373,1374 Activation of TLR-1 and TLR-2 can lead to platelet activation via a GPVI-like mechanism with TLR-4 through the nuclear factor (NF)-κB pathway.1375 All of the components of the LPS signaling complex, including relatively high levels of TLR-41376 and CD14, MD2, and MyD88, have been identified in platelets. LPS binding to platelets stimulates secretion and potentiates agonist-activation by signaling thru the TLR-4 complex.1377 LPS binding to platelet TLR-4 causes release of CD40L1378 and modulates the release of cytokines by platelets.1379,1380 In experimental animal models, TLR-4 may mediate LPS-induced microvascular thrombosis and thrombocytopenia.1376,1381 TLR-4–null mice have prolonged times to vasoocclusion after vascular injury, but endothelial TLR-4 rather than platelet TLR-4 seems to be more important in supporting platelet thrombus formation.1382 The interactions of LPS, produced by toxigenic E. coli, with platelet TLR-4 has been proposed to contribute to the pathophysiology of hemolytic uremic syndrome.1378 Ligand binding to platelet TLR-4 also promotes platelet–neutrophil interactions, neutrophil activation, and along with TLR-2, the formation of NETs, which capture and sequester bacteria from the circulation.792,1383 Activation of TLR-9 with protein adducts leads to Src-dependent platelet activation.1374
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Peroxisome Proliferator-Activated Receptors
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Peroxisome proliferator-activated receptors (PPARs) belong to a nuclear hormone receptor family of ligand-activated transcription factors.1384 PPARγ is one of the three PPAR family members and is widely expressed in white adipose tissue, macrophages, B and T lymphocytes, smooth muscle cells, fibroblasts, and endothelial cells. It has been implicated in metabolism, insulin responsiveness, adipocyte differentiation, immune function, and inflammation. The thiazolidinedione class of insulin-sensitizing drugs used to treat type 2 diabetic patients act by binding PPARγ. Both PPARβ/δ and PPARγ are present in platelets. PPARγ agonists decrease thrombin-induced platelet aggregation and release of ATP, TX, and CD40L.1384 Thus, PPARγ appears to downregulate platelet activation. Activated platelets release PPARγ complexed with the retinoid X receptor.1385 Treatment with select thiazolidinediones has been associated with reductions in markers of platelet activation, including aggregation and P-selectin expression. PPARβ ligands synergize with NO to inhibit platelet function.1386,1387 The antiplatelet effects of the calcium channel blocker nifedipine may be mediated through PPAR receptors.348
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Matrix Metalloproteinases
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Platelets contain a number of MMPs, as well as MMP activators and inhibitors.1388,1389 MMP-1 can be activated by collagen and, in turn, cleave PAR-1 at a site two amino acids N-terminal to the site of thrombin cleavage.1390 This cleavage, like thrombin’s, activates PAR-1 by activating a tethered ligand. Thus, MMP-1 can augment collagen-induced platelet activation mediated by GPVI and integrin α2β1. MMP-2 cleavage has been implicated in enhancing platelet aggregation via cleavage of talin and activation of integrin αIIbβ3.1389 It exists in an inactive form in resting platelets and it is cleaved into its active form when platelets are activated, probably by platelet-type von Willebrand disease (MT1-MMP).1391 It then moves to the surface via binding to integrin αIIbβ3 and may then go on to cleave CD40 ligand.1339 MMP-2 is released into the coronary circulation of patients with acute coronary syndromes.1392 ADAM-17 (TACE) is important in the cleavage of GPIb and the release glycocalicin.1076 MMP-9, which is increased in plasma in models of sepsis, can also cleave platelet CD40L.1393 Other related proteins in platelets include MMP-9 and -14, ADAM-10, and tissue inhibitor of metalloproteinase (TIMP)- 1, -2, and -3. Platelets also contain ADAMTS-13, which cleaves VWF, thus controlling hemostasis and thrombosis (Chap. 126).