title: The role of protein disulfide isomerase in the post-ligation phase of β3 integrin-dependent cell adhesion.
abstract: BACKGROUND
  Protein disulfide isomerase (PDI) catalyzes disulfide bond exchange. It is crucial for integrin-mediated platelet adhesion and aggregation and disulfide bond exchange is necessary for αIIbβ3 and αvβ3 activation. However, the role of disulfide bond exchange and PDI in the post-ligation phase of αIIbβ3 and αvβ3 mediated cell adhesion has yet to be determined.
  
  
  METHODS
  To investigate a possible such role, we expressed wild type (WT) human αIIb and either WT human β3, or β3 harboring single or double cysteine to serine substitutions disrupting Cys473-Cys503 or Cys523-Cys544 bonds, in baby hamster kidney (BHK) cells, leading to expression of both human αIIbβ3 and a chimeric hamster/human αvβ3. Adhesion to fibrinogen-coated wells was studied in the presence or absence of bacitracin, a PDI inhibitor, with and without an αvβ3 blocker.
  
  
  RESULTS
  Flow cytometry showed WT and mutant αIIbβ3 expression in BHK cells and indicated that mutated αIIbβ3 receptors were constitutively active while WT αIIbβ3 was inactive. Both αIIbβ3 and αvβ3 integrins, WT and mutants, mediated adhesion to fibrinogen as shown by reduced but still substantial adhesion following treatment with the αvβ3 blocker. Mutated αIIbβ3 integrins disrupted in the Cys523-Cys544 bond still depended on PDI for adhesion as shown by the inhibitory effect of bacitracin in the presence of the αvβ3 blocker. Mutated integrins disrupted in the Cys473-Cys503 bond showed a similar trend.
  
  
  CONCLUSIONS
  PDI-mediated disulfide bond exchange plays a pivotal role in the post-ligation phase of αIIbβ3-mediated adhesion to fibrinogen, while this step in αvβ3-mediated adhesion is independent of disulfide exchange.
authors.affiliation: Hemostasis Laboratory, Rabin Medical Center, Petah Tikva, Israel; Hematology Institute, Davidoff Cancer Center, Rabin Medical Center, Petah Tikva, Israel; Thrombosis and Hemostasis Unit, Beilinson Hospital, Rabin Medical Center, Petah Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Avi
authors.initials: A
authors.lastname: Leader
authors.affiliation: Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer, Israel.
authors.forename: Ronit
authors.initials: R
authors.lastname: Mor-Cohen
authors.affiliation: Hematology Institute, Davidoff Cancer Center, Rabin Medical Center, Petah Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Ron
authors.initials: R
authors.lastname: Ram
authors.affiliation: Hemostasis Laboratory, Rabin Medical Center, Petah Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Vera
authors.initials: V
authors.lastname: Sheptovitsky
authors.affiliation: Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer, Israel.
authors.forename: Uri
authors.initials: U
authors.lastname: Seligsohn
authors.affiliation: Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer, Israel.
authors.forename: Nurit
authors.initials: N
authors.lastname: Rosenberg
authors.affiliation: Hemostasis Laboratory, Rabin Medical Center, Petah Tikva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address: judith.lahav@gmail.com.
authors.forename: Judith
authors.initials: J
authors.lastname: Lahav
mesh_terms: D000818:Animals; D001414:Bacitracin / Q000737:chemistry; D002384:Catalysis; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000378:metabolism; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D000072417:Protein Domains; D012694:Serine / Q000235:genetics
chemical_list: D004220:Disulfides; D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D001414:Bacitracin; D012694:Serine; D005340:Fibrinogen; D019704:Protein Disulfide-Isomerases; D003545:Cysteine
keywords: Cell adhesion; Disulfide bond; Integrins; PDI; α(IIb)β(3); α(v)β(3)