Identification of von Willebrand factor D4 domain mutations in patients of Afro-Caribbean descent: In vitro characterization.
binding assays
intracellular retention
recombinant proteins
von Willebrand disease
von Willebrand factor
Journal
Research and practice in thrombosis and haemostasis
ISSN: 2475-0379
Titre abrégé: Res Pract Thromb Haemost
Pays: United States
ID NLM: 101703775
Informations de publication
Date de publication:
May 2022
May 2022
Historique:
received:
02
12
2021
revised:
29
03
2022
accepted:
19
04
2022
entrez:
23
6
2022
pubmed:
24
6
2022
medline:
24
6
2022
Statut:
epublish
Résumé
Von Willebrand disease was diagnosed in two Afro-Caribbean patients and sequencing of the VWF gene ( Our goal was to characterize how the D4 variants p.(Pro2145Thrfs*5) and p.(Cys2216Phefs*9) influenced VWF biosynthesis/secretion and functions using in vitro assays. Recombinant VWF (rVWF), mutant or wild-type, was produced via transient transfection of the human embryonic kidney cell line 293T. The use of different tags for the wild-type and the mutant allele allowed us to distinguish between the two forms when measuring VWF antigen in medium and cell lysates. Binding of rVWF to its ligands, collagen, factor VIII, ADAMTS13, and platelet receptors was also investigated. Homozygous expression of the p.(Cys2216Phefs*9)-rVWF mutation resulted in an almost complete intracellular retention of the protein. Heterozygous expression led to secretion of almost exclusively wild-type-rVWF, logically capable of normal interaction with the different ligands. In contrast, the p.(Pro2145Thrfs*5)-rVWF exhibited reduced binding to type III collagen and αIIbβ3 integrin compared to wild-type-rVWF. We report two mutations of the D4 domains that induced combined qualitative and quantitative defects.
Sections du résumé
Background
UNASSIGNED
Von Willebrand disease was diagnosed in two Afro-Caribbean patients and sequencing of the VWF gene (
Objectives
UNASSIGNED
Our goal was to characterize how the D4 variants p.(Pro2145Thrfs*5) and p.(Cys2216Phefs*9) influenced VWF biosynthesis/secretion and functions using in vitro assays.
Methods
UNASSIGNED
Recombinant VWF (rVWF), mutant or wild-type, was produced via transient transfection of the human embryonic kidney cell line 293T. The use of different tags for the wild-type and the mutant allele allowed us to distinguish between the two forms when measuring VWF antigen in medium and cell lysates. Binding of rVWF to its ligands, collagen, factor VIII, ADAMTS13, and platelet receptors was also investigated.
Results
UNASSIGNED
Homozygous expression of the p.(Cys2216Phefs*9)-rVWF mutation resulted in an almost complete intracellular retention of the protein. Heterozygous expression led to secretion of almost exclusively wild-type-rVWF, logically capable of normal interaction with the different ligands. In contrast, the p.(Pro2145Thrfs*5)-rVWF exhibited reduced binding to type III collagen and αIIbβ3 integrin compared to wild-type-rVWF.
Conclusions
UNASSIGNED
We report two mutations of the D4 domains that induced combined qualitative and quantitative defects.
Identifiants
pubmed: 35734101
doi: 10.1002/rth2.12737
pii: S2475-0379(22)01233-X
pmc: PMC9198896
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e12737Informations de copyright
© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).
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