Thromb Haemost 2014; 112(06): 1159-1166
DOI: 10.1160/th14-04-0391
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

Viviana Daidone
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
,
Giovanni Barbon
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
,
Elena Pontara
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
,
Grazia M. Cattini
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
,
Lisa Gallinaro
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
,
Enrico Zampese
2   Department of Biomedical Sciences, Padua, Italy
,
Paola Pizzo
2   Department of Biomedical Sciences, Padua, Italy
,
Alessandra Casonato
1   University of Padua Medical School, Department of Cardiologic, Thoracic and Vascular Sciences, Second Chair of Internal Medicine, Padua, Italy
› Author Affiliations

Financial support: This work was supported in part by a MURST grant (ex 60 % 2011).
Further Information

Publication History

Received: 29 April 2014

Accepted after major revision: 22 July 2014

Publication Date:
18 November 2017 (online)

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Summary

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.