Introduction
Von Willebrand factor (VWF) is a multimeric glycoprotein that plays a
crucial role in primary and secondary hemostasis . Mutations in the VWF
gene (VWF ), causing either quantitative reductions or qualitative
defects of VWF, lead to von Willebrand disease (VWD), the most prevalent
hemorrhagic disorder in humans . VWD is classified into three main
categories. Type 1 and 3 VWD are characterized by mild/moderate
reduction or virtual absence of VWF, respectively, and type 2 VWD is
characterized by functional defects in VWF . VWF is synthesized as a
proVWF containing a 22 amino acid (aa) signal peptide, a 741 aa
propeptide, and a 2050 aa mature VWF molecule. The proVWF is constructed
as repeats in the order: D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK .
VWF monomers are dimerized in the endoplasmic reticulum (ER) by the
formation of disulfide bonds between the C-terminal cysteine-knot (CK)
domains of two monomers . Successively, the proVWF dimers exit the ER
and are transported to the Golgi, where the N-terminal disulfide bonds
between cysteines in the D3 domains lead to the formation of multimers .
The polymerization process is accompanied by proteolytic cleavage of the
VWFpp (domains D1-D2) in the trans-Golgi network (TGN), yielding mature
VWF multimers and propeptide dimers . In the endothelial cells, the VWF
is either secreted directly from the TGN to the plasma membrane, which
is called the constitutive secretion pathway, or it is first stored in
specific organelles, Weibel-Palade bodies (WPBs), and they will be later
released by the regulated exocytosis pathway . Expression of VWF in some
heterologous systems, such as Human embryonic kidney 293 (HEK293) cells,
can drive the formation of WPBs-like organelles (pseudo-WPBs). The VWF
multimers and the propeptide are packaged together to form secretory
WPBs . The VWF propeptide (VWFpp) acts as an intracellular chaperone; it
is required for both the VWF multimerization process and the trafficking
from Golgi to storage vesicles . Furthermore, the propeptide region is
required for the appropriate orientation of the dimeric VWF which is
essential for the VWF helical assembly of tubules in nascent WPBs .
Nonetheless, In the present study, we demonstrated that propeptide
variants disturb the anterograde ER-Golgi trafficking via affecting VWF
recruitment at the ER exit quality control.
In eukaryotic cells, trafficking of newly synthesized proteins is
organized by COPI- and COPII-coated vesicles that contribute to the
recruitment of proteins and ER-Golgi transport, as well as ER exit
quality control (QC) by excluding misfolded cargos from vesicles .
Current studies point to two major protein complexes modulating VWF exit
from ER, ARF (ADP-ribosylation factors) 1-GEF and Sec23/24-Sec22b, which
have a role in selecting and recruitment of the cargo proteins in COPI
and COPII complexes, respectively .
In this study, we investigated the underlying pathophysiological
mechanisms of six novel candidate missense substitutions located in
VWFpp recognized with low VWF secretion and defects in its function in
VWD patients. Four out of the six mutations (p.Gly55Glu, p.Trp191Arg,
p.Asn211Asp, and p.Gly334Glu) were identified as compound heterozygous
in VWD patients with non- typical phenotype (type 2A/2B and type 1/2N
VWD) and type 3 VWD. The other two novel variations (p.Val86Glu and
p.Cys608Trp) were detected as homozygous in type 3 VWD. In the present
study, identified variants were reproduced and heterologously expressed
to monitor intracellular trafficking, multimerization, packaging,
constitutive expression, and binding functions of the VWF mutants.
Furthermore, the putative mechanisms affecting exiting VWF from ER were
evaluated by in silico docking of VWF with COPI and COPII recruiting
proteins. The results of this study enhanced our knowledge about the
biological significance of the VWFpp in health and disease.