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Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.

BACKGROUND Patients with aortic stenosis (AS) can experience bleeding complications including gastrointestinal bleeding from angiodysplastic lesions due to acquired von Willebrand syndrome. Studies have pointed to a role for von Willebrand factor (VWF) in angiogenesis. OBJECTIVE The objective of this study was to assess VWF defects in AS patients over time and the impact on angiogenesis using patient-derived endothelial colony-forming cells (ECFCs). PATIENTS/METHODS Plasma sample collection and ECFC isolations were performed before valve replacement surgery, 3 to 5 days after, and 6 months after surgery. Plasma VWF antigen, activity, propeptide, collagen binding, multimers, factor VIII coagulant activity, and ADAMTS13 activity (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13) were determined. ECFCs were assessed for VWF and angiopoietin-2 (Ang-2) storage and secretion, cell proliferation, and tubule formation in Matrigel. RESULTS AND CONCLUSIONS Aortic stenosis patients exhibited quantitative and qualitative abnormalities of VWF including significantly increased VWF antigen, activity, and propeptide levels following surgery (P

Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

Background 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 Patients and Methods 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. Results 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. Discussion 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. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.