Possible role of protein disulfide isomerase PDIA1 in mechanisms of von Willebrand disease type 3
M. A. Brehm, S. Vollmers, T. Obser, R. Schneppenheim (Hamburg, Germany)
Bleeding disorders - Basic science
Date: 17.02.2017,
Time: 08:00 - 09:15
Objective: The hemostatic activity of von Willebrand factor (VWF) is highly dependent on its presence as a multimeric protein. The biosynthesis of VWF multimers involves formation of multiple disulfide bonds with different functions. First, in the endoplasmic reticulum (ER), intra-monomeric bonds are formed which are responsible for correct folding. Second, formation of bonds between the C-termini of two VWF monomers is catalyzed by protein disulfide isomerase isoform A1 (PDIA1) to produce VWF dimers. Third, multimerization is realized in the Golgi apparatus by formation of N-terminal inter-dimer disulfide bonds catalyzed by the VWF propeptide. Mutations in the VWF gene, which result in lack of VWF in the patient’s plasma, lead to von Willebrand disease (VWD) type 3. Nevertheless, it has been shown that residual VWF may still be produced in the patient’s endothelial cells, which often is retained in the ER. The aim of this study is to determine whether PDIA1 might be involved in this cellular retention of VWF mutants associated with VWD type 3.
Methods: We have investigated eleven VWD type 3 mutants by immunofluorescence after transient expression in HEK293 cells. Parallel staining of endogenous PDIA1 was used to visualize VWF-PDIA1-association and measurement of fluorescence intensities revealed influence of the VWF mutants on PDIA1.
Results: Our preliminary data indicate three different degrees of mutant-VWF-PDIA1-association: 1) The mutants p.Val86Glu, p.Gly160Ala, p.Gly163Val, p.Gly525Glu, and p.Trp1120Ser exhibited normal PDI co-localization compared to wildtype VWF, 2) p.Gly39Arg, p.Leu129Arg and p.Tyr271His showed increased ER localization associated with a slight increase in PDIA1 expression level, 3) The cysteine mutants p.Cys2431Tyr and p.Cys2533Arg induced an alteration in PDIA1 localization that was associated with cluster formation of VWF and PDIA1 in the ER.
Conclusion: While most of the propeptide mutants exhibited ER retention without an effect on PDIA1, three of them induced an increase in PDIA1 expression. The loss of cysteine residues caused formation of VWF-PDIA1 clusters that resulted in a VWF secretion defect and an altered PDIA1 localization and expression. The latter finding indicates that PDIA1 might be involved in mechanisms underlying VWD type 3 by mediating cellular retention of VWF cysteine mutants.