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Storage and secretion of naturally occurring von Willebrand factor A domain variants
Author(s) -
Groeneveld Dafna J.,
Wang JiongWei,
Mourik Marjon J.,
Dirven Richard J.,
Valentijn Karine M.,
Voorberg Jan,
Reitsma Pieter H.,
Eikenboom Jeroen
Publication year - 2014
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/bjh.13074
Subject(s) - von willebrand factor , von willebrand disease , secretion , transfection , mutant , hek 293 cells , endoplasmic reticulum , wild type , mutation , phenotype , point mutation , microbiology and biotechnology , biology , gene , chemistry , medicine , endocrinology , immunology , genetics , platelet
Summary Von Willebrand disease ( VWD ) is a bleeding disorder characterized by reduced plasma von Willebrand factor ( VWF ) levels or functionally abnormal VWF . Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1–A3 domains, that were found in families diagnosed with VWD . Human Embryonic Kidney 293 ( HEK 293) cells were transiently transfected with plasmids encoding full‐length wild‐type VWF or mutant VWF . Six mutations in the A1–A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo‐Weibel‐Palade bodies ( WPB ). In addition, two mutations also showed reduced numbers of pseudo‐ WPB , even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK 293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.