title: C560Rβ3 caused platelet integrin αII b β3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality.
abstract: OBJECTIVE
  β(3)-Deficient megakaryocytes were modified by human β(3)-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ(3) amino acid substitution, for investigation of how an activated αII b β(3) conformation affects platelets in vivo in mice.
  
  
  METHODS
  As in our previous report of an R560β(3) mutation in a patient with Glanzmann thrombasthenia, R560β(3) murine platelets spontaneously bound antibody that only recognizes activated αII b β3 bound to its ligand, fibrinogen.
  
  
  RESULTS
  With this murine model, we showed that αII b -R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β(3) 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560β(3) 'wild-type' mice remained alive. Pathologic examination revealed that R560β(3) mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β(3) megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β(3) washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo.
  
  
  CONCLUSIONS
  The results demonstrate that continuous occupancy of αIIb β3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state.
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: J
authors.initials: J
authors.lastname: Fang
authors.forename: P
authors.initials: P
authors.lastname: Nurden
authors.forename: P
authors.initials: P
authors.lastname: North
authors.forename: A T
authors.initials: AT
authors.lastname: Nurden
authors.forename: L M
authors.initials: LM
authors.lastname: Du
authors.forename: N
authors.initials: N
authors.lastname: Valentin
authors.forename: D A
authors.initials: DA
authors.lastname: Wilcox
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry*; D006470:Hemorrhage / Q000235:genetics; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology; D051379:Mice; D009154:Mutation; D019007:P-Selectin / Q000737:chemistry; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D013577:Syndrome; D013915:Thrombasthenia / Q000235:genetics; D013927:Thrombosis / Q000473:pathology
chemical_list: D039661:Integrin beta3; D019007:P-Selectin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen
keywords: glanzmann thrombasthenia; hematopoietic stem cells; integrin αIIbβ3; lentivirus gene transfer; megakaryocytes; platelets
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