title: Mechanisms of the interaction between two ADAMTS13 gene mutations leading to severe deficiency of enzymatic activity.
abstract: The inherited deficiency of the von Willebrand factor-cleaving protease ADAMTS13 is associated with rare forms of thrombotic thrombocytopenic purpura (TTP). We investigated a woman with a family history of chronic recurrent TTP and undetectable plasma levels of ADAMTS13 activity. Genetic analysis revealed two missense mutations in the heterozygous state: p.Val88Met substitution in the metalloprotease domain and p.Gly1239Val substitution in the first CUB domain of ADAMTS13. To explore the mechanism of ADAMTS13 deficiency in this patient, the wild type (WT; ADAMT13(WT)) and each mutant construct (ADAMTS13(Val88Met), ADAMTS13(Gly1239Val)) were transiently expressed in HEK 293 and COS-7 cells. To recapitulate the compound heterozygous state of the patient, both mutant ADAMTS13 proteins were also expressed together. The p.Val88Met mutation led to a defect of secretion of the protease associated with a reduction of enzymatic activity, the p.Gly1239Val mutation led to a secretion defect causing intracellular accumulation of the protease. The mechanistic effects of the mutations were further explored by means of differential immunofluorescence, that demonstrated an homogeneous distribution of ADAMTS13(WT) in the Cis-Golgi and endoplasmic reticulum (ER) compartments, a reduction of ADAMTS13(Val88Met) in both compartments, while ADAMTS13(Gly1239Val) failed to reach the Cis-Golgi compartment and remained in the ER.
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, IRCCS Maggiore Hospital, Mangiagalli, Regina Elena Foundation and University of Milan, Milan, Italy. flora.peyvandi@unimi.it
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
authors.forename: Silvia
authors.initials: S
authors.lastname: Lavoretano
authors.forename: Roberta
authors.initials: R
authors.lastname: Palla
authors.forename: Carla
authors.initials: C
authors.lastname: Valsecchi
authors.forename: Giuliana
authors.initials: G
authors.lastname: Merati
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: Edoardo
authors.initials: E
authors.lastname: Rossi
authors.forename: Pier
authors.initials: P
authors.lastname: Mannuccio Mannucci
mesh_terms: D051722:ADAM Proteins / Q000172:deficiency* / Q000235:genetics*; D000071120:ADAMTS13 Protein; D000328:Adult; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017077:Culture Media, Conditioned; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human