The main hypothesis of this study is that defects in components of the secretory machinery of VWF are causative for reduced VWF levels and lack of desmopressin response. Also, defects in this machinery may be causative in patients with BUC. To…
ID
Bron
Verkorte titel
Aandoening
Bleeding disorders
Ondersteuning
Onderzoeksproduct en/of interventie
Uitkomstmaten
Primaire uitkomstmaten
1. To establish optimized approaches for the isolation and characterization of BOECs, CD34+-
derived MKs, iPSC- ECs and iPSC-MKs.
2. Dissect the cellular mechanisms that control endothelial and platelet (secretory) function,
thereby understanding the pathophysiology of bleeding disorders and the genetic control of
VWF secretion using BOECs, platelets, CD34+-derived MKs, iPSC- ECs and iPSC-MKs.
3. To develop in vitro methods to introduce or correct (disease-causing) mutations in regulators
of endothelial or platelet function in BOECs or iPSC-derived ECs/MK using CRISPR-mediated
genome engineering.
Achtergrond van het onderzoek
Rationale:
Hemostasis is critically dependent on Von Willebrand factor (VWF), a multimeric
adhesive plasma protein that is crucial for mediating platelet adhesion to sites of vascular
damage and that acts as a chaperone for coagulation factor VIII (FVIII) in plasma. The bulk of
plasma VWF is synthesized by endothelial cells (ECs) where it is stored in and released from
Weibel-Palade bodies (WPBs). Platelets also store and secrete VWF from alpha-granules after
activation, which contributes to thrombus formation. The molecular mechanisms that control
secretion of VWF from ECs and platelets are poorly understood. Abnormalities in the
(secretory) function of platelets and endothelial cells can lead to bleeding, such as observed
in platelet storage pool disorders (SPD), Von Willebrand disease (VWD) or in individuals with
“low VWF”.
Problem definition: In ~30% of type 1 VWD patients and individuals with low VWF (defined
as VWF levels <50 IU/dL) no VWF mutations are found. Another problem is that in around 50%
of bleeding patients referred to tertiary centers the underlying mechanisms that cause clinically
relevant bleeding problems cannot yet be identified, so-called bleeding of unknown cause
(BUC). Desmopressin is administered therapeutically to improve platelet function and raise
VWF in plasma by inducing WPB exocytosis from endothelial cells. In ~25% of patients
desmopressin fails to trigger (sufficient) release of VWF/FVIII for reasons largely unknown.
Research hypothesis: The main hypothesis of this study is that defects in components of the
secretory machinery of VWF are causative for reduced VWF levels and lack of desmopressin
response. Also, defects in this machinery may be causative in patients with BUC. To identify
new determinants of VWF levels and vascular health we will study secretory mechanisms in
ECs and platelets of individuals with abnormalities in hemostatic and/or secretory function. For
this we will establish ex vivo (patient-derived) cellular model systems of endothelial and platelet
secretion using blood outgrowth endothelial cells (BOECs), platelets, CD34+-derived
megakaryocytes and induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs)
and megakaryocytes (iPSC-MKs).
Doel van het onderzoek
The main hypothesis of this study is that defects in components of the
secretory machinery of VWF are causative for reduced VWF levels and lack of desmopressin
response. Also, defects in this machinery may be causative in patients with BUC. To identify
new determinants of VWF levels and vascular health we will study secretory mechanisms in
ECs and platelets of individuals with abnormalities in hemostatic and/or secretory function. For
this we will establish ex vivo (patient-derived) cellular model systems of endothelial and platelet
secretion using blood outgrowth endothelial cells (BOECs), platelets, CD34+-derived
megakaryocytes and induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs)
and megakaryocytes (iPSC-MKs).
Onderzoeksopzet
A maximum of seventy milliliters of blood will be drawn for the isolation of PBMCs,
platelets, plasma and DNA. Several parameters will be measured in plasma to characterize
the hemostatic and angiogenic profile. BOECs, iPSC-ECs/MKs and CD34+-derived primary
MKs will be characterized in vitro. Protein expression profiles will be determined by whole
proteome mass spectrometry. DNA will be genotyped for SNPs and mutations in VWF and
VWF associated genes.
Onderzoeksproduct en/of interventie
The intervention in this study is venous blood sampling. A maximum of seventy milliliters of blood will be drawn for the isolation of PBMCs,
platelets, plasma and DNA.
Publiek
Wetenschappelijk
Belangrijkste voorwaarden om deel te mogen nemen (Inclusiecriteria)
Patients
Prior diagnosis of VWD, low VWF, inherited platelet function defects, bleeding of unknown
cause or genetic disorders in which dysfunction of the secretory pathway has been
implicated in disease pathogenesis.
For patients ≥16 years old; written informed consent
For patients 12-15 years old; written informed consent from both the patient and their
parent(s)/legal guardian(s)
For patients <12 years old; written informed consent from their parent(s)/legal guardian(s)
Family members
Age 18 years or older
Family member of a patient with VWD, low VWF, inherited platelet function defects,
bleeding of unknown cause or genetic disorders in which dysfunction of the secretory
pathway has been implicated in disease pathogenesis.
Family members may or may not be affected.
Healthy controls
Age 18 years or older
Not previously diagnosed with VWD, low VWF, inherited platelet function defects, bleeding
of unknown cause or genetic disorders in which dysfunction of the secretory pathway has
been implicated in disease pathogenesis.
Belangrijkste redenen om niet deel te kunnen nemen (Exclusiecriteria)
A potential subject who meets any of the following criteria will be excluded from participation
in this study:
Unable to give written informed consent.
Use of medication that can compromise platelet function or hemostasis.
Opzet
Deelname
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In overige registers
| Register | ID |
|---|---|
| NTR-new | NL8923 |
| Ander register | METC Erasmus MC : MEC-2020-0214 |