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Integrin αIIbβ3 outside-in signaling

Tom N. Durrant, Marion T. van den Bosch and Ingeborg Hers

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    The early stages of αIIbβ3outside-in signaling. (A) Following inside-out signaling, the integrin adopts a conformation that enables it to bind ligands such as fibrinogen with high affinity. c-Src can associate with the RGT motif of the β3-integrin C-terminal tail via its SH3 domain. (B) Integrin clustering supports full c-Src activation, bringing distinct c-Src proteins into proximity for trans-autophosphorylation. For the sake of clarity, all subsequent figures depict nonclustered αIIbβ3. (C) Src supports the activation of Syk kinase, which may bind via its SH2 domains to the β3 C-terminal tail in a manner independent of β3 tyrosine phosphorylation, or to phosphorylated tyrosines within the ITAM of FcγRIIa. (D) αIIbβ3-mediated platelet aggregation leads to SFK-mediated phosphorylation of tyrosine residues within the NxxY motfis of the β3-integrin C-terminal tail, leading to the recruitment of proteins such as Grb2, Shc, and myosin. Hashed lines represent phosphorylation events, denoted on proteins by a yellow circle.

  • Figure 2.

    Outside-in signaling downstream of SFKs and Syk. SFKs phosphorylate a host of enzymes and signaling adaptors downstream of activated αIIbβ3, which are important for processes such as platelet spreading. These include PLCγ2, FAK, and ADAP, which in turn further propagate signal transduction. PLCγ2 catalyzes the formation of DAG and IP3 from membrane PtdIns(4,5)P2, leading to PKC activation and calcium liberation, respectively. PKCβ and PKCθ can localize to the β3-integrin tail via RACK1. FAK activation can be supported by CIB-1 bound to the αIIb C-terminal tail, and FAK substrates include the actin-binding protein α-actinin. Syk kinase phosphorylates further downstream targets, including SLP-76, and Vav-family RhoGEFs, which interplay with SFK substrates to propagate outside-in signaling. Hashed lines represent phosphorylation events, denoted on proteins by a yellow circle. Syk may also associate directly with the β3-integrin C-terminal tail.

  • Figure 3.

    Outside-in signaling through class I PI3Ks. Class I PI3Kβ is particularly important for thrombus stability, and is activated downstream of αIIbβ3 via a pathway involving the kinases Src, Syk, and Pyk2. This leads to phosphorylation of the E3-protein ubiquitin ligase c-Cbl, which associates with the p85 regulatory subunit of class I PI3K. Activated class I PI3Ks phosphorylate membrane PtdIns(4,5)P2 to form PtdIns(3,4,5)P3, which leads to the recruitment and/or activation of a range of PtdIns(3,4,5)P3-binding proteins. These include kinases such as BTK/Tec, PDK1, and AKT. PtdIns(3,4,5)P3 can also regulate a range of GAPs and GEFs for small GTPases, including RASA3, dedicator of cytokinesis (DOCK) proteins, and Cytohesin-family members. Syk may also associate directly with the β3-integrin C-terminal tail. Hashed lines represent phosphorylation events; yellow circles represent activating phosphorylation; orange circles represent inhibitory phosphorylation.

  • Figure 4.

    Outside-in signaling to the actomyosin cytoskeleton via Rho-family small GTPases. The Rho GTPases are particularly important for platelet spreading and retraction. (A) The activation status of the 3 Rho-family small GTPases, Rac, Cdc42, and RhoA is regulated by GAPs and GEFs downstream of activated integrins. When GTP-bound and active, these small GTPases signal to the actomyosin cytoskeleton via multiple effector proteins. Cdc42 and Rac can promote Arp2/3-mediated actin polymerization via WASP and WAVE proteins, respectively, whereas RhoA promotes MLC phosphorylation via ROCK-mediated inhibition of MLC phosphatase (MLCP). These small GTPases may also regulate actin dynamics via proteins such as cofilin, and via formins. (B) In platelets, regulation of RhoA activity coordinates platelet spreading and subsequent clot retraction, as discussed in panels i and ii. Hashed lines represent phosphorylation events; yellow circles represent activating phosphorylation; orange circles represent inhibitory phosphorylation.

  • Figure 5.

    Direct αIIbβ3-cytoskeletal coupling. A number of proteins permit direct coupling of integrins to the actin cytoskeleton, which is important in platelets for processes such as clot retraction. However, the current understanding of this coupling is limited in platelets relative to other cell types. Talin can provide a direct link between the β3-integrin C-terminal tail and actin, and has been reported to be important for clot retraction. Stretch-induced changes in talin lead to the exposure of binding sites for vinculin, although the role for this protein in platelet αIIbβ3 signaling may be minimal. Paxillin and α-actinin can associate with the αIIbβ3 C-terminal tails, and may regulate integrin affinity and actin coupling. Kindlin-3 can couple directly to β3 integrins, and to the actin cytoskeleton via the heterotrimeric complex of ILK, PINCH, and Parvin. ILK can itself couple directly to β3 integrins, and also acts as a scaffold to recruit further proteins. Myosin can bind directly to the tyrosine-phosphorylated β3 C-terminal tail. It is important to note that the integrin-binding sites for many of the depicted proteins may overlap (see “Proteins enabling more direct integrin-cytoskeleton coupling” and Figure 6). The αIIbβ3 adhesome is likely to involve a number of further proteins permitting direct coupling of the integrin to the actin cytoskeleton, which are yet to be identified.

  • Figure 6.

    The β3C-terminal tail serves as a docking site for multiple proteins involved in integrin signaling. Shown are the reported binding sites for the indicated proteins. The 2 NxxY motifs of the β3 tail are underlined, with the phosphorylatable tyrosine residues in bold.

Tables

  • Table 1.

    Key proteins involved in αIIbβ3 outside-in signaling in platelets

    ProteinKnockout mouse platelet phenotype (unless otherwise stated)Reference
    Positive regulators
     ADAPReduced attachment and spreading on fibrinogen under shear flow. Unstable thrombi. Increased tail rebleeding.48, 52
     c-CblImpaired spreading on fibrinogen. Delayed clot retraction.98
     CD148Reduced spreading on fibrinogen. Compromised thrombus formation and stability. Increased tail bleeding.64
     Cdc42Conflicting reports regarding role in filopodia formation on fibrinogen. Accelerated arterial occlusive thrombus formation but prolonged bleeding times.113, 114
     CIB1Reduced spreading on fibrinogen. Impaired arterial occlusion associated with unstable thrombus formation. Increased tail-bleeding time.87, 155
     Dab2Impaired spreading on fibrinogen. Impaired thrombus formation. Impaired clot retraction. Prolonged bleeding time.156
     FAKImpaired spreading on fibrinogen. Increased tail rebleeding.82
     Gα13Impaired stable thrombus formation. Increased tail-bleeding time. Contrasting reports regarding role in spreading on fibrinogen. Mutation of the Gα13-binding β3 ExE motif, or peptide-mediated inhibition of the β3-Gα13 interaction, inhibited platelet spreading on fibrinogen and thrombus formation, whereas the peptide did not affect tail bleeding.43, 63, 102, 157
     ILKImpaired thrombus stability. Increased tail bleeding time and volume.135, 136
     Kindlin-3Severe bleeding during development. Loss of spreading on fibrinogen. Loss of thrombus formation. Increased tail bleeding.133
     LnkImpaired spreading on fibrinogen. Reduced thrombus stability. Impaired clot retraction. Increased tail bleeding.137
     PDK1Reduced spreading on fibrinogen. Reduced thrombus formation. Delayed clot retraction.101
     PECAM-1Reduced spreading on fibrinogen. Delayed clot retraction.158
     PI3Kβp110β KO, kinase-dead, and pharmacological inhibition: reduced spreading on fibrinogen. Almost complete inability to adhere to fibrinogen under flow. Unstable thrombi. Delayed clot retraction. No effect on rodent tail-bleeding time.91-93, 95
     PKCPKCθ; reduced spreading on fibrinogen yet enhanced thrombus formation due to enhanced secretion. PKCα; reduced thrombus formation due to reduced secretion. PKCβ; reduced spreading.78, 79, 159, 160
     PLCγ2Defective spreading on fibrinogen.76
     Pyk2Defective spreading on fibrinogen. Impaired thrombus formation. Slightly prolonged tail-bleeding time.97, 161
     Rac1Defective spreading on fibrinogen. Reduced thrombus formation and stability. Prolonged tail bleeding.109-111
     Rasa3Rasa3 RapGAP activity restrains Rap1-driven cell spreading on fibrinogen.47
     RhoANormal extent of spreading on fibrinogen but with slightly altered morphology. Required for thrombus stability. Essential for clot retraction. Increased tail bleeding.102
     ROCK2Impaired thrombus formation. Prolonged tail-bleeding time.162
     SLP-76Fetal hemorrhage and platelet dysfunction including impaired spreading on fibrinogen.75, 84, 85
     SFKsDeletion of the c-Src–docking sequence in β3 impacts platelet spreading on fibrinogen and impairs thrombus formation and stability. These mice also show increased tail bleeding. Mouse platelets deficient in c-Src display impaired spreading on fibrinogen. Some redundancy with other SFKs such as Fyn and Lyn occurs, whereas Lyn is important for thrombus formation. However, Lyn also holds a negative regulatory role in cell spreading. Loss of SFKs does not affect tail bleeding. Mutation of Tyr 747 and Tyr 759 residues in the β3-integrin tail, which are phosphorylated by SFKs, leads to unstable platelet aggregates, impaired clot retraction, and increased tail rebleeding.55-57, 65, 69
     SykImpaired spreading on fibrinogen.55
     Tetraspanin CD151Impaired spreading on fibrinogen. Delayed clot retraction. Moderate in vivo bleeding defect.143
     Tetraspanin TSSC6Impaired spreading on fibrinogen. Impaired thrombus stability. Impaired clot retraction. Increased tail bleeding.142
     Vav1/3Impaired spreading on fibrinogen.77
     VPS33BImpaired spreading on fibrinogen. Impaired stable thrombus formation. Impaired clot retraction. Increased tail bleeding.106
     WASPWAS patients and WASP KO mice: Decreased spreading on fibrinogen. Impaired clot retraction. Increased tail rebleeding.117
    Negative regulators
     Dok-1Increased spreading on fibrinogen after thrombin stimulation. Accelerated thrombus formation. Increased clot retraction. Shortened bleeding time.138
     Dok-2Shear-dependent increase in αIIbβ3 adhesive function with accelerated thrombus growth in vivo.139
     JAM-AEnhanced spreading on fibrinogen. Enhanced thrombus formation. Enhanced clot retraction. Shortened tail-bleeding time.163
     PaxillinKnock down: Increased spreading on fibrinogen. Enhanced thrombus formation. Enhanced clot retraction. Reduced tail-bleeding time.131
     SHIP1Conflicting reports from 2 mouse lines. One study reports enhanced spreading on fibrinogen, and enhanced adhesion and spreading on fibrinogen under flow due to enhanced stability of adhesive contacts. In contrast, another study reports defects in thrombus formation, increased tail-bleeding time, and impaired clot retraction due to a role for SHIP1 in platelet contractility and thrombus organization.11, 164
     Tetraspanin CD82Enhanced clot retraction. Reduced bleeding time.165
    • Presented are the phenotypes of platelets with deficiency or inhibition of a range of proteins involved in the αIIbβ3 outside-in signaling pathway, with a focus on the key outside-in driven processes of platelet spreading, stable thrombus formation, and clot retraction. Bleeding is also included for consideration of whether proteins may be potential candidates for the therapeutic targeting of the αIIbβ3 outside-in signaling pathway to prevent unwanted thrombosis, while avoiding unwanted bleeding.

    • JAM-A, junctional adhesion molecule-A; PECAM-1, platelet endothelial cell adhesion molecule-1.