- Hamideh Yadegari1,
- Julia Driesen1,
- Anna Pavlova1,
- Arijit Biswas1,
- Vytautas Ivaskevicius1,
- Robert Klamroth2 and
- Johannes Oldenburg1,*
- 1 Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany;
- 2 Haemophilia Treatment Center, Vivantes Hospital Friedrichshain, Berlin, Germany
- ↵* Corresponding author; email:
The carboxyl-terminal domains of von Willebrand factor, D4-CK, are cysteine rich implying their structural importance. The present study characterized the impact of five cysteine missense mutations residing in D4-CK domains on conformation and biosynthesis of von Willebrand factor. These variants were identified as heterozygous in type 1 (p.Cys2619Tyr and p.Cys2676Phe), type 2A (p.Cys2085Tyr and p.Cys2327Trp) and as compound heterozygous in type 3 (p.Cys2283Arg) von Willebrand disease. Transient expression of human cell lines with wild-type or mutant von Willebrand factor constructs was performed. Quantitative and qualitative assessment of mutated recombinant von Willebrand factor was done in comparison with recombinant wild-type von Willebrand factor. Storage of von Willebrand factor in pseudo-Weible-Palade bodies was studied with confocal microscopy. Structural impact of the mutations was analyzed by homology modeling. Homozygous expressions showed that these mutations caused defects in multimerization, elongation of pseudo-Weible-Palade bodies and secretion of von Willebrand factor. Co-expressions of wild-type von Willebrand factor and p.Cys2085Tyr, p.Cys2327Trp and p.Cys2283Arg demonstrated defect in multimer assembly, suggesting a new pathologic mechanism for dominant type 2A von Willebrand disease due to mutations in D4 and B domains. Structural analysis revealed that mutations p.Cys2283Arg, p.Cys2619Tyr and p.Cys2676Phe disrupted intra-domain disulfide bonds, whereas p.Cys2327Trp might affect an inter-domain disulfide bond. The p.Cys2327Trp variant is distinguished from the other mutants by an electrophoretic mobility shift of the multimer bands. The results highlight the importance of cysteine residues within the C-terminal of von Willebrand factor on structural conformation of protein and consequently multimerization, storage, and secretion of von Willebrand factor.
- Received January 14, 2013.
- Accepted March 20, 2013.
- Copyright © 2013, Ferrata Storti Foundation