Establishment of Blood Outgrowth Endothelial Cells from a Type 3 Von Willebrand Disease (VWD) Patient with a Neutralizing Anti-VWF Inhibitors

Background: Type 3 von Willebrand disease (VWD) is characterized by a complete loss type of von Willebrand Factor (VWF) with the rarest disease frequency and hemorrhagic symptom is the most severe among other VWD types. The development of alloantibodies directed against VWF occurs in approximately 10% of patients with type 3 VWD. In this study, we found the VWF gene alterations and established blood outgrowth endothelial cells (BOECs) a from a Japanese type 3 VWD patient with an anti-VWF inhibitors.

Case: A 5-year-old woman suffered from epistaxis, purpura and easy bruising and has been diagnosed as VWD. Her plasma level of FVIII:C was 1.8% and VWF:Ag and VWF:RCo was <5% and <10%, respectively. Her plasma lacked VWF multimers, indicating that she had type 3 VWD. To treat her frequent muco-cutaneous bleeding, purified plasma-derived FVIII/VWF concentrates were administrated followed by inhibitor development. After about 20ED of FVIII/VWF concentrates, an anaphylactic symptom comprised of cough, dyspnea, and wheezing developed at the time of dosing. An high titer inhibitory antibody against VWF was confirmed by a Bethesda assay based on a assay of VWF:RCo. Her younger sister also had type 3 VWD.

Methods: Patients samples were collected after the written informed consent has bee obtained. We performed MLPA for genetic deletion or insertion. Then we amplified the all exons including the exon/intron boundaries of the VWF gene by PCR using allele-specific primers, and analyzed DNA sequences of the patient. Peripheral blood mononuclear cells were obtained from the patient and BOECs were then established. Briefly, buffy coat mononuclear cells were isolated and seeded onto a 6-well tissue culture plate precoated with type 1 rat tail collagen at 37°C, 5% CO2, in a humidified incubator. Medium was changed daily for 7 days and then every other day until the first passage. The endothelial identity of the BOECs was confirmed by flow cytometric and immunofluorescence analyses using endothelial markers with antibodies to CD31, VE-Cadherin (CD144) and VEGFR-2 (CD309).

Results: The large deletion and insertion of VWF gene were not found. Direct sequencing showed the propositus and her sister were compound heterozygous for an E2341X (c.7021G>T) mutation in exon41 and a Y2631X (c.7892-7893insA) mutation in exon48. These were therefore two novel nonsense mutations and normal VWF polypeptides could not be translated. Flow cytometric analysis indicated that established BOECs expressed cell surface CD31 and CD309, whereas CD34 was not detected. By immunofluorescence analysis of fixed BOECs, VWF signal was remarkably reduced from the patient, in spite of normal VE-Cadherin expression. VWF specific ELISA was performed for both cell supernatant and lysates of patient’s BOECs but immunodetectable VWF was not secreted.

Discussion: We identified two novel nonsense mutations causing type3 VWD. BOECs established from the patient reproduced the phenotype of the disease, suggesting that BOEC analysis is useful for studying the molecular pathogenesis of von Willebrand disease.