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2606 Characterization and Quantification of Von Willebrand Factor Proteoforms in Von Willebrand Disease Patients and Healthy Controls: Insights from the Win-Study

Program: Oral and Poster Abstracts
Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Translational Research
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Iris charlotte Kreft1*, Tirsa T van Duijl2*, Calvin Van Kwawegen3*, Winny Phan1*, Margo Schuller4*, Carmen van Der Zwaan2*, Alexander Meijer, PhD4*, Ruben Bierings5*, Frank W.G. Leebeek, MD, PhD3 and Maartje Van Den Biggelaar4*

1Sanquin Research, Amsterdam, Netherlands
2Sanquin Research, amsterdam, Netherlands
3Department of Haematology, Erasmus University Medical Center-Erasmus MC, Rotterdam, Rotterdam, Netherlands
4Sanquin Research, Amsterdam, NLD
5Erasmusmc, University Medical Center Rotterdam, Rotterdam, NLD


Von Willebrand disease (VWD) is a commonly inherited bleeding disorder which is characterized by either low (type 1) or absent (type 3) levels of Von Willebrand factor (VWF) or by the occurrence of dysfunctional VWF proteoforms (type 2) in plasma. The complexity of diagnosing and managing VWD arises from limited understanding of the diverse VWF proteoforms present in both healthy individuals as well as in VWD patients.

Objective: The aim of this study was to investigate the plasma proteome and to characterize VWF proteoforms in Von Willebrand disease (VWD) patients, that were enrolled in the Willebrand in the Netherlands (WiN)1 study, compared with healthy controls.


We selected type 1, type 2A, type 2M, type 2N and type 3 patients from the WiN cohort for which extensive laboratory- and genotype data were available. Plasma from 64 VWD patients and ten healthy controls was analyzed using a combination of discovery- and targeted mass spectrometry-based approaches.


Both discovery-based as well as targeted quantitative proteomics analysis revealed significant variation in VWF levels across the selected VWD patient group, which was in line with reported clinical laboratory data. Elevated VWF-propeptide/VWF ratios were observed in plasma samples from a range of VWD patients, with notable increases observed in VWD type 2A, B, and M, indicating accelerated clearance of these VWF proteoforms. Discovery-based proteomics revealed a strong VWF-F8 relationship in all VWD subtypes and healthy controls, except for type 2N VWD. We were able to identify peptides corresponding with common polymorphisms (e.g. p.Thr798Ala, p.Gln852Arg, p.Thr1381Ala), for which the observed peptide distribution was in line with the minor allele frequency reported by the Broad institute. In addition, we identified peptides corresponding with 16 pathogenic VWF variants (e.g. p.Arg1306Trp, p. Phe1293Leu, p.Cys1190Arg, p.Arg1374His or p.Tyr1584Cys) of which p.Cys1190Arg wild-type and variant peptide aligned with the zygosity at DNA level (Figure X). Based on spike-in with stable isotope-labeled peptides, the variant-to-wildtype ratios varied among VWD patients with distinct pathogenic variants, ranging from 0.5-2.5 in type 2M (p.Phe1293Leu) and type 1 patients (p.Tyr1584Cys), <1 in type 2A patients (p.Arg1374His), to >8 in type 2B patients (p.Arg1306Trp), suggesting distinct stoichiometry of VWF variants among these patients (Figure 2).


Our study highlights the feasibility of VWF proteoform identification and quantification from plasma and suggest a potential role in unravelling their contribution to the biological variations and clinical diversity observed in VWD.

1 De Wee et al., Determinants of bleeding phenotype in adult patients with moderate or severe von Willebrand disease. Thromb Haemost. 2012 Oct;108(4):683-92.

Disclosures: Leebeek: CSL Behring, Takeda, UniQure: Consultancy, Research Funding; Sobi: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Biomarin: Consultancy.

*signifies non-member of ASH