title: Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3.
abstract: OBJECTIVE
  Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown.
  
  
  RESULTS
  Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA.
  
  
  CONCLUSIONS
  PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom. c.i.jones@reading.ac.uk.
authors: Chris I
authors: CI
authors: Jones
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Tanya
authors: T
authors: Sage
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Leonardo A
authors: LA
authors: Moraes
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Sakthivel
authors: S
authors: Vaiyapuri
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Umara
authors: U
authors: Hussain
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Katherine L
authors: KL
authors: Tucker
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Natasha E
authors: NE
authors: Barrett
authors: From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, United Kingdom.
authors: Jonathan M
authors: JM
authors: Gibbins
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D034281:Dynamins / Q000502:physiology*; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D053778:Thiazolidines / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000494:pharmacology*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D053778:Thiazolidines; D014841:von Willebrand Factor; D015638:Cytochalasin D; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D013917:Thrombin; D034281:Dynamins; C037067:latrunculin A; D002118:Calcium
keywords: blood platelets; glycoproteins; signal transduction; thrombin; von Willebrand factor