von Willebrand factor is dimerized by protein disulfide isomerase

Abstract

Key Points• The protein disulfide isomerase is involved in VWF dimerization by initiating disulfide bond formation at cysteines 2771 and 2773.• von Willebrand diseaseassociated mutations in the dimerization domain of von Willebrand factor disturb processing by the protein disulfide isomerase.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 K D 5 236 6 66 nM and K D 5 282 6 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar K D (258 6 104 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 Cys2771-27739 and Cys27719-2773. Subsequently, the third bond, Cys2811-28119, 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. (Blood.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

“…In line with prior studies linking P-selectin and C3 activation, 12 blocking the P-selectin ligand PSGL-1 similarly reduced platelet and fibrin deposition (supplemental Figure 3). Because platelet activation 47,49 and the platelet ligand VWF are regulated by thioldisulfide exchange reactions [50][51][52] and C5 conversion, but not further downstream complement components, and is known to alter the redox state of cell surface PDI, 18,53 we compared the inhibitory effects of C3 deficiency with inhibition of PDI by PACMA31. PACMA31 has an optimal fit with the second catalytic domain of PDI, 54,55 and this PDI domain has recently been shown to be crucial for regulating platelet activation and thrombosis in vivo.…”

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA

“…Dimerization of VWF takes place in the endoplasmic reticulum (ER), after which VWF multimers are formed in the Golgi. 22 To test whether VWF is retained in the ER, we performed a co-staining of VWF and ER marker PDI. Indeed, a clear overlap of VWF and PDI staining is observed in ECFC 2A transfected with siNEG, indicating ER retention (►Fig.…”

Alles fließt?

“…Die beteiligten Cysteine wurden durch die kristallographische Strukturaufklärung der CK‐Domäne bestimmt . Mithilfe umfangreicher biochemischer und biophysikalischer Untersuchungen konnte das Enzym, welches die Ausbildung der Disulfidbrücken katalysiert, als Proteindisulfidisomerase (PDI) Isoform A1 identifiziert werden . Den ersten Hinweis auf die PDI erbrachte die Kolokalisation mit VWF‐Molekülen in der Größenordnung eines VWF‐Dimers im ER von Endothelzellen.…”