Endothelial Colony-Forming Cells in Patients With VWD, AVWS and Healthy Subjects (ECFCs/2022)

Isolation and Characterization of Endothelial Colony Forming Cells (ECFCs) in Patients Diagnosed With Von Willebrand Disease, Acquired Von Willebrand Syndrome and Healthy Subjects

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.

Study Overview

• Status: Recruiting
• Conditions: Von Willebrand Disease (VWD); Acquired Von Willebrand Disease
• Intervention / Treatment: Diagnostic test: Blood sample collection for VWF measurements in plasma; Other: Blood sample collection for ECFC isolation and characterization; Other: Genetic testing of VWF

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 48

Contacts and Locations

• Study Contact: Name: Flora Peyvandi, MD, PhD; Phone Number: +39 02-55035414; Email: flora.peyvandi@policlinico.mi.it

Study Locations

• Italy
  • Milan, Italy, 20122
    • Recruiting
    • Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, A.B.Bonomi Hemophilia and Thrombosis Center
    • Contact: Flora Peyvandi, MD, PhD; Phone Number: +39 02-55035414; Email: flora.peyvandi@policlinico.mi.it

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

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.

Description

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.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
von willebrand disease/ Acquired von Willebrand disease patients; Patients with a confirmed diagnosis of VWD or AVWS. Participants will undergo peripheral blood sample collection for plasma VWF measurements and ECFCs isolation and characterization. In patients with VWD without prior molecular characterization, an additional blood sample will be collected for genetic analysis.	Diagnostic test: Blood sample collection for VWF measurements in plasma; Plasma samples will be collected for the measurement of VWF levels.; Other: Blood sample collection for ECFC isolation and characterization; Blood samples will be collected to isolate ECFCs and perform their subsequent characterization.; Other: Genetic testing of VWF; An additional blood sample will be collected for VWF genetic testing in patients with VWD without prior molecular characterization.
Healthy donors; Healthy volunteers with no personal or family history of bleeding or thrombotic disorders, serving as the reference population for the study. Participants will undergo peripheral blood sample collection plasma VWF measurements and ECFCs isolation and characterization.	Diagnostic test: Blood sample collection for VWF measurements in plasma; Plasma samples will be collected for the measurement of VWF levels.; Other: Blood sample collection for ECFC isolation and characterization; Blood samples will be collected to isolate ECFCs and perform their subsequent characterization.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
ECFCs isolation and characterization	Assessment of differences between ECFCs derived from patients with VWD or AVWS and healthy controls. Basal and stimulated VWF production and secretion by ECFCs, including basal VWF levels in culture media and cell lysates and stimulated VWF release following exposure to biological or chemical compounds, measured by ELISA (IU/dL) and compared between patients with VWD/AVWS and healthy controls. VWF mRNA expression levels in ECFCs, assessed by quantitative real-time PCR and expressed as ΔΔCt, compared between patients with VWD/AVWS and healthy controls.	42 weeks from enrollment start

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Correlation of ECFC VWF Antigen Protein and mRNA Levels with Plasma and Platelet VWF Antigen/Activity Levels and Molecular Defect.	Correlation analyses between ECFC-derived VWF parameters and circulating VWF levels in corresponding patients with VWD, AVWS, and healthy controls. ECFC-derived VWF antigen levels in culture media and cell lysates (IU/dL), measured by ELISA, and VWF mRNA expression levels in ECFCs (ΔΔCt), assessed by quantitative real-time PCR, will be correlated with plasma and platelet VWF antigen and activity levels (IU/dL). All ECFC-derived and circulating VWF parameters will additionally be analyzed in relation to the specific underlying molecular defect.	42 weeks from enrollment start
Comparison of angiogenic properties of ECFCs from patients with VWD/AVWS and healthy controls	Angiogenic properties of ECFCs will be assessed using an in vitro Matrigel tube formation assay and compared between patients with type 2 or type 3 VWD or AVWS and healthy controls. Quantitative parameters will include total tube length (μm), number of tubes, number of branches, and number of meshes.	42 weeks from enrollment start
Evaluation of Allele-Specific siRNA to Restore Normal VWF Levels in Type 2A VWD	Allele-specific small interfering RNA (siRNA) will be evaluated in endothelial ECFCs derived from a patient with type 2A VWD to assess whether selective inhibition of the mutant allele restores normal VWF expression and secretion. All analyses will be performed before and after siRNA transfection. The following parameters will be assessed: VWF mRNA expression levels, assessed by quantitative real-time PCR and expressed as ΔΔCt (relative expression); VWF protein levels in ECFC culture media, measured by ELISA (IU/mL).	42 weeks from enrollment start

Collaborators and Investigators

• Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

Study record dates

Study Major Dates

• Study Start (Actual): November 11, 2023
• Primary Completion (Estimated): December 31, 2026
• Study Completion (Estimated): May 31, 2028

Study Registration Dates

• First Submitted: December 17, 2025
• First Submitted That Met QC Criteria: January 13, 2026
• First Posted (Actual): January 22, 2026

Study Record Updates

• Last Update Posted (Actual): January 22, 2026
• Last Update Submitted That Met QC Criteria: January 13, 2026
• Last Verified: December 1, 2025

More Information

Terms related to this study

• Keywords: von Willebrand factor; von Willebrand disease; ex vivo; endothelial colony forming cells (ECFCs)
• Additional Relevant MeSH Terms: Genetic Diseases, Inborn; Hematologic Diseases; Blood Coagulation Disorders; Hemorrhagic Disorders; Blood Platelet Disorders; Blood Coagulation Disorders, Inherited; Coagulation Protein Disorders; Congenital, Hereditary, and Neonatal Diseases and Abnormalities; Hemic and Lymphatic Diseases; von Willebrand Diseases
• Other Study ID Numbers: 3387 n.6569

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No