title: Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis.
abstract: BACKGROUND
  Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin.
  
  
  OBJECTIVE
  To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin.
  
  
  METHODS
  We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparin-affinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer.
  
  
  RESULTS
  We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop.
  
  
  CONCLUSIONS
  Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.
authors.affiliation: Department of Medicine, University of Murcia, Murcia, Spain. jcc@um.es
authors.forename: J
authors.initials: J
authors.lastname: Corral
authors.forename: J A
authors.initials: JA
authors.lastname: Huntington
authors.forename: R
authors.initials: R
authors.lastname: González-Conejero
authors.forename: A
authors.initials: A
authors.lastname: Mushunje
authors.forename: M
authors.initials: M
authors.lastname: Navarro
authors.forename: P
authors.initials: P
authors.lastname: Marco
authors.forename: V
authors.initials: V
authors.lastname: Vicente
authors.forename: R W
authors.initials: RW
authors.lastname: Carrell
mesh_terms: D000328:Adult; D000368:Aged; D000990:Antithrombin III / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D020152:Antithrombin III Deficiency / Q000150:complications / Q000235:genetics*; D004252:DNA Mutational Analysis; D019281:Dimerization; D004220:Disulfides; D005192:Family Health; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010375:Pedigree; D011487:Protein Conformation; D015843:Serpins / Q000737:chemistry / Q000235:genetics; D013030:Spain; D020246:Venous Thrombosis / Q000097:blood / Q000235:genetics*
chemical_list: D004220:Disulfides; D015843:Serpins; D000990:Antithrombin III