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

Roberta Donadelli, Federica Banterla, Miriam Galbusera, Cristina Capoferri, Sara Bucchioni, Sara Gastoldi, Silvia Nosari, Giuseppe Monteferrante, Zaverio M. Ruggeri, Elena Bresin, Friedrich Scheiflinger, Edoardo Rossi, Constantino Martinez, Rosanna Coppo, Giuseppe Remuzzi, Marina Noris

Research output: Contribution to journalArticle

Abstract

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), I nonsense (W1016Stop) and I 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 the V88M 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 activit, 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 ADAMTS 13 mutations.

Original languageEnglish
Pages (from-to)454-464
Number of pages11
JournalThrombosis and Haemostasis
Volume96
Issue number4
DOIs
Publication statusPublished - Oct 2006

Fingerprint

Purpura
Mutation
Thrombotic Thrombocytopenic Purpura
Peptide Hydrolases
Drosophila
Single Nucleotide Polymorphism
Drosophila Proteins
HEK293 Cells
von Willebrand Factor
Protein Sorting Signals
In Vitro Techniques
ADAMTS13 Protein
Introns
Exons
Thrombosis
Antigens
Genes

Keywords

  • ADAMTS13
  • Expression studies
  • Mutations
  • Thrombotic thrombocytopenic purpura

ASJC Scopus subject areas

  • Hematology

Cite this

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic trombocytopenic purpura. / Donadelli, Roberta; Banterla, Federica; Galbusera, Miriam; Capoferri, Cristina; Bucchioni, Sara; Gastoldi, Sara; Nosari, Silvia; Monteferrante, Giuseppe; Ruggeri, Zaverio M.; Bresin, Elena; Scheiflinger, Friedrich; Rossi, Edoardo; Martinez, Constantino; Coppo, Rosanna; Remuzzi, Giuseppe; Noris, Marina.

In: Thrombosis and Haemostasis, Vol. 96, No. 4, 10.2006, p. 454-464.

Research output: Contribution to journalArticle

Donadelli, R, Banterla, F, Galbusera, M, Capoferri, C, Bucchioni, S, Gastoldi, S, Nosari, S, Monteferrante, G, Ruggeri, ZM, Bresin, E, Scheiflinger, F, Rossi, E, Martinez, C, Coppo, R, Remuzzi, G & Noris, M 2006, 'In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic trombocytopenic purpura', Thrombosis and Haemostasis, vol. 96, no. 4, pp. 454-464. https://doi.org/10.1160/TH06-05-0236
Donadelli, Roberta ; Banterla, Federica ; Galbusera, Miriam ; Capoferri, Cristina ; Bucchioni, Sara ; Gastoldi, Sara ; Nosari, Silvia ; Monteferrante, Giuseppe ; Ruggeri, Zaverio M. ; Bresin, Elena ; Scheiflinger, Friedrich ; Rossi, Edoardo ; Martinez, Constantino ; Coppo, Rosanna ; Remuzzi, Giuseppe ; Noris, Marina. / In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic trombocytopenic purpura. In: Thrombosis and Haemostasis. 2006 ; Vol. 96, No. 4. pp. 454-464.
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AU - Galbusera, Miriam

AU - Capoferri, Cristina

AU - Bucchioni, Sara

AU - Gastoldi, Sara

AU - Nosari, Silvia

AU - Monteferrante, Giuseppe

AU - Ruggeri, Zaverio M.

AU - Bresin, Elena

AU - Scheiflinger, Friedrich

AU - Rossi, Edoardo

AU - Martinez, Constantino

AU - Coppo, Rosanna

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AU - Noris, Marina

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