title: Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction.
abstract: BACKGROUND
  Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.
  
  
  METHODS
  In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.
  
  
  RESULTS
  Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.
  
  
  CONCLUSIONS
  Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.
authors.affiliation: The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Israel. Electronic address: ronit.cohen@sheba.health.gov.il.
authors.forename: Ronit
authors.initials: R
authors.lastname: Mor-Cohen
authors.affiliation: The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Israel.
authors.forename: Nurit
authors.initials: N
authors.lastname: Rosenberg
authors.affiliation: The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Israel.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Averbukh
authors.affiliation: The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Sackler Faculty of Medicine, Tel Aviv University, Israel.
authors.forename: Uri
authors.initials: U
authors.lastname: Seligsohn
authors.affiliation: Hemostasis Laboratory, Rabin Medical Center, Petah Tiqva, Israel; Sackler Faculty of Medicine, Tel Aviv University, Israel.
authors.forename: Judith
authors.initials: J
authors.lastname: Lahav
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D003018:Clot Retraction / Q000502:physiology*; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D004220:Disulfides; D039302:Integrin alphaVbeta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005337:Fibrin
keywords: Clot retraction; Disulfide bonds; Integrins; Thiols; αIIbβ3; αvβ3