Title: von Willebrand factor is dimerized by protein disulfide isomerase
Authors: Lippok, Svenja ×
Kolšek, Katra
Löf, Achim
Eggert, Dennis
Vanderlinden, Willem
Müller, Jochen P
König, Gesa
Obser, Tobias
Röhrs, Karoline
Schneppenheim, Sonja
Budde, Ulrich
Baldauf, Carsten
Aponte-Santamaría, Camilo
Gräter, Frauke
Schneppenheim, Reinhard
Rädler, Joachim O
Brehm, Maria A #
Issue Date: Mar-2016
Publisher: W.B. Saunders
Series Title: Blood vol:127 issue:9
Article number: 10.1182/blood-2015-04-641902
Abstract: Multimeric von Willebrand factor (VWF) is essential for primary hemostasis. The biosynthesis of VWF high-molecular-weight multimers requires spatial separation of each step because of varying pH value requirements. VWF is dimerized in the endoplasmic reticulum by formation of disulfide bonds between the C-terminal cysteine knot (CK) domains of 2 monomers. Here, we investigated the basic question of which protein catalyzes the dimerization. We examined the putative interaction of VWF and the protein disulfide isomerase PDIA1, which has previously been used to visualize endoplasmic reticulum localization of VWF. Excitingly, we were able to visualize the PDI-VWF dimer complex by high-resolution stochastic optical reconstruction microscopy and atomic force microscopy. We proved and quantified direct binding of PDIA1 to VWF, using microscale thermophoresis and fluorescence correlation spectroscopy (dissociation constants KD = 236 ± 66 nM and KD = 282 ± 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar KD (258 ± 104 nM) measured for PDI interaction with the isolated CK domain and the atomic force microscopy images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. On the basis of protein-protein docking and molecular dynamics simulations, combined with fluorescence microscopy studies of VWF CK-domain mutants, we suggest the following mechanism of VWF dimerization: PDI initiates VWF dimerization by forming the first 2 disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond, Cys2811-2811', is formed, presumably to protect the first 2 bonds from reduction, thereby rendering dimerization irreversible. This study deepens our understanding of the mechanism of VWF dimerization and the pathophysiological consequences of its inhibition.
ISSN: 0006-4971
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Non-KU Leuven Association publications
× corresponding author
# (joint) last author

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