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

Abstract

Background von Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra‐molecular chaperone for mature VWF in sorting to its storage organelles, Weibel‐Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype. Objective To investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient‐derived endothelial colony‐forming cells (ECFCs). Patients/Methods Endothelial colony‐forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4‐5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected). Results Endothelial colony‐forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide‐isomerase (PDI), indicating an ER‐retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non‐null variants. Conclusion This study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient‐derived cells.

Current Knowledge of Enterococcal Endocarditis: A Disease Lurking in Plain Sight of Health Providers

“…This process is believed to inhibit platelet recruitment and other responses of the host coagulation cascade by shrouding the bacteria in host vWF. Or, conversely, a previous paper report by Gaytán et al [ 181 ] showed that a new adhesin that binds to sialic acid is crucial for infective endocarditis in several bacterial species. However, it is unclear how this relates to enterococcal endocarditis.…”

A transcriptome analysis of basal and stimulated VWF release from endothelial cells derived from patients with type 1 VWD

“…VWF, VE-Cadherin, and nuclear staining was performed as described previously. 19 The coverslips were then mounted to slides using antifade mounting medium (Vecta-Sheild) and imaged using a Leica TCS SP8 confocal fluorescence microscope and the LASX software (Leica Microsystems). Images were taken by a blinded microscopist.…”

“…Confocal immunofluorescence microscopy: For confocal immunofluorescence (IF) microscopy, the stimulation protocol was performed as described above with the exception that collagen coated coverslips were added to the wells and cells seeded to these. VWF, VE-Cadherin, and nuclear staining was performed as described previously 19 . The coverslips were then mounted to slides using anti-fade mounting medium (Vecta-Sheild) and imaged using a Leica TCS SP8 confocal fluorescence microscope and the LASX software (Leica Microsystems).…”

Potentials of Endothelial Colony-Forming Cells: Applications in Hemostasis and Thrombosis Disorders, from Unveiling Disease Pathophysiology to Cell Therapy

“…The data obtained from these studies led to the discovery the novel pathogenesis mechanisms leading to VWF deficiencies in VWD patients and clarifying the effect of VWF variants on intracellular processing of VWF, including multimerization, storage/formation of WPBs, and secretion. 22,[44][45][46][47][48][49][50][51] The use of the patient-derived ECFCs emphasized the significance of the exonic synonymous and deep intronic variants on VWF splicing and their contribution to the pathogenesis of VWD. [44][45][46][47]51 Furthermore, we inspected the gene expression profile and trafficking of the inflammatory/angiogenesis proteins Ang2 and P-selectin, co-stored with VWF in WPBs, in the patient-and healthy control-derived ECFCs.…”