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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 due to varying pH value requirements. VWF is dimerized in the endoplasmic reticulum (ER) by formation of disulfide bonds between the CK domains of two monomers. Here, we investigated the basic question 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 ER localization of VWF. Excitingly, we were able to visualize the PDI-VWF-dimer-complex by high-resolution STORM and Atomic Force Microscopy (AFM). We proved and quantified direct binding of PDIA1 to VWF using Microscale Thermophoresis (MST) and Fluorescence Correlation Spectroscopy (FCS) (dissociation constants K_D = 236±66 nM and K_D = 282±123 nM by MST and FCS, respectively). The similar K_D (258±104 nM) measured for PDI interaction with the isolated CK domain and the AFM images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. Based on 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 two disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond Cys2811-2811' is formed, presumably, to protect the first two 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.