von Willebrand factor propeptide missense variants affect anterograde transport to Golgi resulting in ER retention

Abstract

von Willebrand disease (VWD), the most prevalent congenital bleeding disorder, arises from a deficiency in von Willebrand factor (VWF), which has crucial roles in hemostasis. The present study investigated functional consequences and underlying pathomolecular mechanisms of several VWF propeptide (VWFpp) missense variants detected in our cohort of VWD patients for the first time. Transient expression experiments in HEK293T cells demonstrated that four out of the six investigated missense variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) severely impaired secretion. Their cotransfections with the wild‐type partly corrected VWF secretion, displaying loss of large/intermediate multimers. Immunostaining of the transfected HEK293 cells illustrated the endoplasmic reticulum (ER) retention of the VWF variants. Docking of the COP I and COP II cargo recruitment proteins, ADP‐ribosylation factor 1 and Sec24, onto the N‐terminal VWF model (D1D2DʹD3) revealed that these variants occur at VWFpp putative interfaces, which can hinder VWF loading at the ER exit quality control. Furthermore, quantitative and automated morphometric exploration of the three‐dimensional immunofluorescence images showed changes in the number/size of the VWF storage organelles, Weibel‐Palade body (WPB)‐like vesicles. The result of this study highlighted the significance of the VWFpp variants on anterograde ER‐Golgi trafficking of VWF as well as the biogenesis of WPB‐like vesicles.

von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient‐derived endothelial colony‐forming cells

“…The authors of this study postulate that hindering glycosylation at this site may affect the ability of VWF to interact with the correct chaperone pathway and thus results in misfolding 30 . A recent study of VWF propeptide missense variants transiently transfected into HEK293T cells illustrated similar cellular findings to ours and showed through modelling that anterograde ER to Golgi transport of VWF was hindered, resulting in ER retention 34 …”

“…30 A recent study of VWF propeptide missense variants transiently transfected into HEK293T cells illustrated similar cellular findings to ours and showed through modelling that anterograde ER to Golgi transport of VWF was hindered, resulting in ER retention. 34 Whether the accumulation in the ER of our VWF variants is a result of hindered chaperone interactions or failure to be effectively trafficked to the Golgi remains to be determined.…”

Genetic Alterations, DNA Methylation, Alloantibodies and Phenotypic Heterogeneity in Type III von Willebrand Disease

“…These mutations (particularly p.V86E by affecting structured H-bonding) are predicted to influence the maturation, multimerization and secretion of VWF protein. Indeed, p.V86E demonstrated severely impaired secretion of mutant VWF protein, suggesting its intracellular retention in transient expression experiments using HEK293T cells [ 34 ]. Similarly, VWF protein multimers are formed in the Golgi due to the N-terminal disulfide bonding between cysteines in the D3 domains [ 35 ].…”

“…Among these, the case of p.P1266L is particularly interesting. Although analysis of the resolved crystal structure of the VWF A1 domain localizes p.P1266L mutation to a loop region with minimal resultant conformational changes in domain structure, p.P1266L has previously been reported as a gain of function variant with enhanced VWF binding to platelets GPIb and resulting in type-2B vWD [ 34 ]. Altogether, the reported pathogenic mutants may cause their deleterious effects by influencing various steps in VWF biogenesis or physiology, manifesting ultimately as vWD.…”

A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel–Palade Bodies

“…In the ECs, VWF is stored in EC-specific secretory organelle, Weibel–Palade bodies (WPBs). The primary product of VWF undergoes modifications including dimerization (through CK domains), multimerization (by disulfide bonds between D3 domains), and proteolytic cleavage of the propeptide, followed by tubular packing of multimers and targeting into WPBs [ 11 , 12 , 13 , 14 , 15 ]. WPBs are elongated storage/secretory organelles that contain VWF, a prerequisite for WPB biogenesis, as well as inflammatory and proangiogenic molecules such as P-selectin and angiopoietin-2 (Ang2) [ 16 , 17 , 18 , 19 ].…”