Integrin αIIbβ3 outside-in signaling

Abstract

Integrin α_IIbβ₃ is a highly abundant heterodimeric platelet receptor that can transmit information bidirectionally across the plasma membrane, and plays a critical role in hemostasis and thrombosis. Upon platelet activation, inside-out signaling pathways increase the affinity of α_IIbβ₃ for fibrinogen and other ligands. Ligand binding and integrin clustering subsequently stimulate outside-in signaling, which initiates and amplifies a range of cellular events driving essential platelet processes such as spreading, thrombus consolidation, and clot retraction. Integrin α_IIbβ₃ has served as an excellent model for the study of integrin biology, and it has become clear that integrin outside-in signaling is highly complex and involves a vast array of enzymes, signaling adaptors, and cytoskeletal components. In this review, we provide a concise but comprehensive overview of α_IIbβ₃ outside-in signaling, focusing on the key players involved, and how they cooperate to orchestrate this critical aspect of platelet biology. We also discuss gaps in the current understanding of α_IIbβ₃ outside-in signaling and highlight avenues for future investigation.

Figure 1.

The early stages of α_IIbβ₃outside-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 α_IIbβ₃. (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) α_IIbβ₃-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 α_IIbβ₃, 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 α_IIb 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 α_IIbβ₃ 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 α_IIbβ₃-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 α_IIbβ₃ signaling may be minimal. Paxillin and α-actinin can associate with the α_IIbβ₃ 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 α_IIbβ₃ 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.