PTP-1B is an essential positive regulator of platelet integrin signaling
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.
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[Show abstract] [Hide abstract] ABSTRACT: Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.
- "Remarkably, we demonstrated a significant effect on collagen contraction. Higher contractile capacity of PTP1B-deficient cells may explain defects in clot retraction in platelets (Salgado et al., 2005), cell migration in fibroblasts (Hernández et al., 2006; Burdisso et al., 2013), axon elongation (Fuentes and Arrequi, 2009), and dendritic spine maturation (Fuentes et al., 2012). "
[Show abstract] [Hide abstract] ABSTRACT: During clot formation, platelets are subjected to various different signals and cues as they dynamically interact with extracellular matrix proteins such as von Willebrand factor (vWF), fibrin(ogen) and collagen. While the downstream signaling of platelet-ligand interactions is well-characterized, biophysical cues, such as hydrodynamic forces and mechanical stiffness of the underlying substrate, also mediate these interactions and affect the binding kinetics of platelets to these proteins. Recent studies have observed that, similar to nucleated cells, platelets mechanosense their microenvironment and exhibit dynamic physiologic responses to biophysical cues. This review discusses how platelet mechanosensing is affected by the hydrodynamic forces that dictate vWF-platelet interactions and fibrin polymerization and network formation. The similarities and differences in mechanosensing between platelets and nucleated cells and integrin-mediated platelet mechanosensing on both fibrin(ogen) and collagen are then reviewed. Further studies investigating how platelets interact with the mechanical microenvironment will improve our overall understanding of the hemostatic process. Copyright © 2015. Published by Elsevier Ltd.
- "While Src is involved in the downstream signaling of the GPIb–A1 interaction , it also constitutively associates with the cytoplasmic tails of β3 and is essential to αIIbβ3 outside-in signaling in platelets. Upon fibrinogen ligation, integrin clustering causes protein tyrosine phosphatase (PTP) 1B-dependent dissociation of c-terminal Src kinase (Csk) from cytoplasmic tails of β3 [98,99], which in turn activates Src. Activated Src is found to localize in filopodia and at the edges of spreading platelets. "
[Show abstract] [Hide abstract] ABSTRACT: The coagulation process is activated by tight control mechanisms, in which platelets play prominent and unique roles. In thrombosis and hemostasis, activated platelets regulate the coagulation system in various ways: by exposing a phosphatidylserine surface for thrombin formation, by supporting fibrin formation, and by regulating the retraction of a fibrin clot. In this review we discuss the involvement of platelet receptors, other membrane proteins, downstream signaling proteins, cytoskeleton-linked proteins and plasma proteins in these procoagulant functions. Studies with both genetically modified mice and pharmacological inhibitors indicate that, for collagen-adhered platelets, in part common signaling pathways lead to phosphatidylserine exposure, generation of thrombin and fibrin, and retraction of the fibrin clot. However, prolonged Ca(2+) elevation leads to thrombin generation, whereas integrin-dependent signaling stimulates fibrin clot retraction. Contact-dependent signaling pathways, triggered by homotypic platelet-platelet interactions, act in particular via the integrin route. © 2014 Elsevier Ltd. All rights reserved.
- "* PI3 kinase LY-294002 CR ↓  * PI3 kinase β Pik3cb-null, TGX-221 CR ↓  Protein phosphatase 1 Pp1cg -/- CR ↓  PTP1B Ptp1b -/- CR = / ↓ [100, 101] Cytoskeleton linked proteins Rac1 EHT-1864 CR ↓  Rap1b Rap1b -/- CR ↓  RhoA Rhoa -/- CR ↓  Talin-1 Tln1-null CR ↓  WASP Wasp -/- CR ↓  Platelet signaling proteins (other) * Calpain Capn1 -/- CR ↓  Cbl Cbl -/-, Cbl (LOF) CR ↓  Gas6 Gas6 -/- TG =  Lnk Sh2b3 -/- CR ↓  mTOR rapamycin CR ↓  NF-κB BAY-117082 CR ↓  PER2 Per2-null CR ↓  Phosphodiesterase 3 milrinone TG ↓  * Phospholipase Cγ2 Plcg -/- CR ↓  SHIP1 (PIP 3 phosphatase) Inpp55-null CR ↓  Plasma proteins * Factor XII F12 -/-, CTI TG ↓  * Factor XI F11 -/- TG ↓  * Factor VII FVIIai TG ↓  * (Pro)thrombin melagatran TG ↓  "