The goal of this observational study is to learn how endothelial colony-forming cells (ECFCs) behave in people with von Willebrand disease (VWD), acquired von Willebrand syndrome (AVWS), and in healthy individuals.

The study aims to establish a reference population of endothelial colony-forming cells (ECFCs) from healthy subjects and compare them with ECFCs derived from patients with VWD/AVWS.

This comparison will help identify cellular and molecular alterations underlying qualitative and quantitative VWF defects. Findings will be correlated with clinical and biochemical data to clarify disease mechanisms.

Study Design: This is a national, monocentric, non-pharmacological study promoted by Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico and coordinated by SC Medicina - Emostasi e Trombosi.

Enrollment: Patients with a prior diagnosis of VWD or AVWS referred to the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center will be contacted for scheduled enrollment. Historical clinical, biochemical, and molecular data will be reviewed, informed consent obtained, and a study-specific blood sample collected to confirm VWF levels and isolate ECFCs.

Healthy volunteers with no history of bleeding or thrombotic disorders will be enrolled in a number equal to that of the patient group. After a brief health interview and informed consent, they will undergo the same blood collection procedures as patients.

Sample collection:

Participants will undergo blood withdrawal for the following procedures:

• Eight milliliters of plasma in sodium citrate will be aliquoted and stored at -80 °C. These samples will be used to confirm patients' diagnoses and, for controls, to measure VWF levels.
• Genetic analysis: one citrate tube will be collected for congenital patients without previous molecular characterization.
• A total of 50 ml of blood in lithium heparin will be drawn for ECFC isolation following the previously published protocol. The isolation procedure will be performed within 3 h of sample collection. In case of unsuccessful isolation, participants may be asked to undergo a second and final blood draw.

Plasma sample testing common to all enrolled subjects: all enrolled subjects will undergo standard plasma testing, including VWF antigen, FVIII activity, VWF activity (VWF:RCo or VWF:GPIbR), VWF collagen-binding activity, and VWF propeptide.

Low-resolution VWF multimer analysis will be performed using either the in-house method or a the semi-automated system.

Procedure to be Performed on ECFCs (common to all enrolled subjects):

• ECFC isolation will follow a modified version of the protocol by Martin-Ramirez et al. (Nat Protoc. 2012;7:1709-1715).
• Flow cytometry will be performed to confirm endothelial lineage.
• Confocal immunofluorescence microscopy will be performed by plating ECFCs on rat tail collagen type I-coated glass slides. Selected markers will include DAPI for nuclei, VWF, and other endothelial markers.
• Basal and stimulated VWF secretion (after histamine or PMA exposure) will be assessed by measuring VWF:Ag levels in culture media and cell lysates using an in-house ELISA.
• VWF multimeric analysis will be performed using the same procedure applied to plasma samples.
• RNA expression analysis will include RNA isolation, first-strand cDNA synthesis, and quantitative real-time PCR for VWF and additional markers using pre-designed TaqMan® assays (ThermoFisher Scientific), normalized to GAPDH or other housekeeping genes.

Assay limited to ECFCs isolated from group B patients and selected controls:

Angiogenesis assay will be performed using Matrigel (Corning). ECFCs obtained from healthy volunteers will be used to establish the assay conditions and will serve as a reference for the analysis of ECFCs isolated from patients.

Assay limited to ECFCs isolated form group C and selected controls: sub-group study:

Inhibition of the mutant allele using small interfering RNA (siRNA) will be performed in a patient selected based on the specific genetic defect (type 2A mutation). ECFCs from this patient will be transfected with a customized siRNA targeting the mutant allele, while a commercial VWF-specific siRNA and a negative control siRNA will be used as controls on both type 2A ECFCs and healthy volunteer-derived ECFCs. All ECFC procedures will be repeated after siRNA silencing, and results will be compared with those obtained from healthy control ECFCs.

Data analysis Continuous variables will be reported as median and interquartile range, whereas categorical variables will be presented as counts and percentages. Laboratory results will be expressed as mean and standard deviation. Comparisons will be performed using one-way ANOVA or Kruskal-Wallis tests, depending on whether variables follow a normal or non-normal distribution. A p-value <0.05 will be considered statistically significant.

Correlation analyses between VWF mRNA expression levels and VWF secreted in cell media, cell lysates, and corresponding plasma VWF levels will be conducted using Pearson's or Spearman's rank correlation, as appropriate.

When available, analyses will be performed using more than one ECFC clone from each enrolled subject.

Inclusion Criteria for patients:

Patients with von Willebrand disease (VWD) or acquired von Willebrand syndrome (AVWS)

Age ≥ 16 years.

Previous diagnosis of von Willebrand disease or acquired von Willebrand syndrome, defined as one of the following:

Group A - Type 1 VWD:

VWF levels ≤ 30 IU/dL, regardless of bleeding history, or

VWF levels ≤ 0.50 IU/mL in the presence of abnormal bleeding.

Group B - Congenital or acquired VWD (VWD or AVWS):

Diagnosis of congenital or acquired VWD, with or without gastrointestinal bleeding.

Group C - Subgroup study (Type 2A VWD):

One patient with type 2A VWD selected for a dedicated sub-study involving allele-specific siRNA silencing of the mutant allele.

Ability and willingness to provide written informed consent.

For patients without prior molecular characterization: willingness to undergo VWF gene sequencing and to sign the related informed consent.

Inclusion criteria for healthy volunteers

• No prior diagnosis of VWD, bleeding disorders, or thrombotic disorders.
• Willingness to donate blood for study procedures.
• Ability and willingness to provide written informed consent.
• Age ≥ 18 years.

Exclusion criteria for both patients and healthy volunteers:

• Pregnancy.
• Anemia, as determined at screening or based on medical history.

The study will include the enrollment of subjects with a previous diagnosis of VWD or AVWS and a group of healthy volunteers (controls). Patients will be selected among those referred at Angelo Bianchi Bonomi Haemophilia and Thrombosis Center, with a previous diagnosis of VWD or AVWS.

Healthy volunteers, defined as individuals without a previous diagnosis of bleeding or thrombotic disorders will be enrolled in equal number to cases with the aim of creating a ECFCs reference group. At enrollment, controls will undergo an interview to confirm their health status, they will be asked to sign the informed consent and then they will undergo a blood withdrawal necessary to confirm plasma VWF levels and to isolate ECFCs as already described for patients.