Heterogeneity Of Type 3 Von Willebrand Disease (VWD): Evidence From Patient-Derived Blood Outgrowth Endothelial Cells (BOEC)

Type 3 von Willebrand Disease (VWD) is the rarest and most severe form of the disease. Patients exhibit plasma von Willebrand factor (VWF) levels of<5% for both VWF:Ag and VWF:RCo and have low FVIII levels (<10%). Patient-derived blood outgrowth endothelial cells (BOEC) provide a useful cellular model for studying VWD instead of using heterologous cell systems, which do not completely mimic the native environment. Until recently, we believed the lack of endogenous VWF in patients with type 3 VWD might pose as a challenge for isolating and culturing type 3 VWD BOEC. The objectives of the current study were to isolate BOEC from type 3 VWD patients with different genotypes and to compare the cellular presence, if any, and localization of VWF in the patient-derived BOEC using confocal immunofluorescence microscopy.

Peripheral blood samples were obtained from seven type 3 VWD index cases (IC) (from six different families) in order to isolate and culture BOEC. These patients were originally enrolled in the Canadian type 3 VWD study as IC or siblings of IC. Research Ethics Board approval was obtained, as well as informed consent from all participants. Endothelial cell phenotype of isolated cells was confirmed using flow cytometry for cell surface markers; positive for CD31, CD144 and/or CD146, and negative for CD14 and CD45. Confocal immunofluorescence microscopy was used to observe intracellular VWF.

We were able to successfully isolate and culture BOEC from seven type 3 VWD patients with different genotypes. All cells were confirmed as endothelial cells as per the markers listed in the methods. Table 1 shows the patient characteristics, and corresponding VWF phenotype and genotype.

Despite a virtual lack of VWF in the plasma of the seven type 3 VWD patients, confocal immunofluorescence microscopy studies showed different patterns of intracellular VWF. Qualitative and/or quantitative defects in Weibel-Palade bodies (WPB) were observed. T001, T076, and T076-S, IC whom are homozygous for VWF propeptide mutations, show a complete lack of WPB formation, with only diffuse staining of VWF, indicative of retention in the endoplasmic reticulum (ER). This was confirmed by co-localization of the diffuse VWF with the ER marker calnexin. T105 and T154, who have in common a frameshift mutation in the CK domain of VWF showed similar VWF staining patterns with diffuse staining and a limited number of rounded WPBs. T151, a compound heterozygote for a nonsense mutation in exon 28 and a splice site mutation in intron 34, showed both diffuse VWF staining, as well as a number of rounded WPBs. The latter three cases indicate abnormalities in WPB biogenesis which would impair secretion of VWF. T050, who is compound heterozygous for two different VWF propeptide mutations, showed a complete absence of VWF, presenting a true VWF “null” endothelial cell.

We have shown that BOEC can be successfully isolated from type 3 VWD patients with different VWF genotypes. As well, we have further confirmed the heterogeneity in type 3 VWD as seen in the variety of endothelial cellular phenotypes associated with the different VWF genotypes. Patient-derived BOEC are a useful cellular model for investigating the pathogenic nature of VWF mutations reflective of the native vascular environment. As well, they may be a useful tool to study the effect of novel treatments for VWD, leading the way for personalized treatment of VWD.

James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.