Thromb Haemost 2006; 96(04): 454-464
DOI: 10.1160/TH06-05-0236
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura

Roberta Donadelli
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Federica Banterla
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Miriam Galbusera
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Cristina Capoferri
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Sara Bucchioni
2  Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy
,
Sara Gastoldi
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
,
Silvia Nosari
2  Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy
,
Giuseppe Monteferrante
2  Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy
,
Zaverio M. Ruggeri
3  Roon Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
,
Elena Bresin
2  Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy
,
Friedrich Scheiflinger
4  Baxter BioScience, Biomedical Research Center, Orth/Donau, Austria
,
Edoardo Rossi
5  Immunohematology and Blood Transfusion Service, Luigi Sacco Hospital, Milan, Italy
,
Constantino Martinez
6  Centro Regional de Hemo-donación, University of Murcia, Spain
,
Rosanna Coppo
7  Department of Nephrology, Dialysis and Transplantatiuon, Regina Margherita Hospital, Turin, Italy
,
Giuseppe Remuzzi
1  Mario Negri Institute for Pharmacological Research, Bergamo, Italy
8  Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy
,
Marina Noris
2  Clinical Research Center for Rare Diseases, Aldo e Cele Daccò, Villa Camozzi-Ranica, Italy
,
the International Registry of Recurrent and Familial HUS/TTP› Institutsangaben
Financial support: This work was supported by grants # 2003.1666/10.4963 from Fondazione Cassa di Risparmio delle Provincie Lombarde, #526/A22 from Istituto Superiore di Sanità Progetto Malattie Rare and GGP02162 from Telethon.
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Publikationsverlauf

Received 03. Mai 2006

Accepted after revision 16. August 2006

Publikationsdatum:
29. November 2017 (online)

Summary

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.