title: Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor.
abstract: Cysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients' plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted - apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients' plasma VWF. The in vitro expression of the E244Lfs*211-VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.
authors.forename: V
authors.initials: V
authors.lastname: Daidone
authors.forename: G
authors.initials: G
authors.lastname: Barbon
authors.forename: E
authors.initials: E
authors.lastname: Pontara
authors.forename: G M
authors.initials: GM
authors.lastname: Cattini
authors.forename: L
authors.initials: L
authors.lastname: Gallinaro
authors.forename: E
authors.initials: E
authors.lastname: Zampese
authors.forename: P
authors.initials: P
authors.lastname: Pizzo
authors.affiliation: A. Casonato, Via Ospedale Civile 105, 35128 Padova, Italy, Tel.: +39 049 821 7177, Fax: +39 049 657391, E-mail: sandra.casonato@unipd.it.
authors.forename: A
authors.initials: A
authors.lastname: Casonato
mesh_terms: D000328:Adult; D000368:Aged; D019943:Amino Acid Substitution; D017077:Culture Media, Conditioned / Q000378:metabolism; D003545:Cysteine; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006487:Hemostasis / Q000235:genetics*; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D010641:Phenotype; D010649:Phenylalanine; D055503:Protein Multimerization; D021381:Protein Transport; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000378:metabolism; D014842:von Willebrand Diseases / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
chemical_list: D017077:Culture Media, Conditioned; D014841:von Willebrand Factor; D010649:Phenylalanine; D003545:Cysteine
keywords: VWF gene mutations; VWF multimerisation; VWF propeptide; von Willebrand disease; von Willebrand factor