title: The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice.
abstract: BACKGROUND
  Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP.
  
  
  OBJECTIVE
  To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo.
  
  
  METHODS
  All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor.
  
  
  RESULTS
  We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor.
  
  
  CONCLUSIONS
  Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.
authors.affiliation: Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: E
authors.initials: E
authors.lastname: De Cock
authors.forename: C
authors.initials: C
authors.lastname: Hermans
authors.forename: J
authors.initials: J
authors.lastname: De Raeymaecker
authors.forename: K
authors.initials: K
authors.lastname: De Ceunynck
authors.forename: B
authors.initials: B
authors.lastname: De Maeyer
authors.forename: N
authors.initials: N
authors.lastname: Vandeputte
authors.forename: A
authors.initials: A
authors.lastname: Vandenbulcke
authors.forename: H
authors.initials: H
authors.lastname: Deckmyn
authors.forename: H
authors.initials: H
authors.lastname: Rottensteiner
authors.forename: M
authors.initials: M
authors.lastname: De Maeyer
authors.forename: S F
authors.initials: SF
authors.lastname: De Meyer
authors.forename: K
authors.initials: K
authors.lastname: Vanhoorelbeke
mesh_terms: D051722:ADAM Proteins / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D000071120:ADAMTS13 Protein; D000328:Adult; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000097:blood; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005260:Female; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006720:Homozygote; D006801:Humans; D008666:Metalloendopeptidases / Q000172:deficiency / Q000235:genetics*; D018345:Mice, Knockout; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D010641:Phenotype; D011247:Pregnancy; D011485:Protein Binding; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000201:enzymology / Q000235:genetics*; D014162:Transfection
chemical_list: D051722:ADAM Proteins; C502939:ADAMTS13 protein, mouse; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D002118:Calcium
keywords: ADAMTS13 protein, human; ADAMTS13 protein, mouse; animal model; mutation; thrombotic thrombocytopenic purpura