PB0819 - Quantification of von Willebrand factor proteoforms by mass spectrometry-based proteomics reveals differential variant-to-wild type stoichiometry in subtypes of Von Willebrand Disease

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Tirsa T. van Duijl, PhD (she/her/hers)

Postdoctoral fellow
Sanquin Research
Amsterdam, Noord-Holland, Netherlands

Co-Author(s)

IK

Iris C. Kreft

PhD student
Sanquin Research
Amsterdam, Noord-Holland, Netherlands
Calvin B. van Kwawegen, MD (he/him/his)

PhD-candidate
Department of Hematology, Erasmus MC, Erasmus University Medical Center Rotterdam, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
Ferdows Atiq, MD, MSc, PhD

Senior Research Fellow
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
Dublin, Dublin, Ireland
WP

Winny Phan

MSc student
Sanquin Research
Amsterdam, Noord-Holland, Netherlands
MS

Margo B.P. Schuller

Bioinformatician
Sanquin Research
Amsterdam, Noord-Holland, Netherlands
Cv

Carmen van der Zwaan

Technician
Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
Amsterdam, Noord-Holland, Netherlands
AM

Alexander B. Meijer

PI
Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
Arie J. Hoogendijk, PhD

PI
Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands.
Amsterdam, Noord-Holland, Netherlands
Ruben Bierings, PhD

Principal investigator
Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
Frank W.G. Leebeek, MD, PhD

Professor and Head of Department of Hematology
Erasmus MC, University Medical Center Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Mv

Maartje van den Biggelaar, PhD

PI
Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands

Background: Von Willebrand Disease (VWD) is the most common 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 proteoforms of VWF (type 2). The relationship between genotypic variation, quantitative levels of VWF and its proteoforms in the circulation and bleeding phenotype is complex and largely unknown.

Aims: We here aim to quantify VWF, VWF propeptide (VWFpp) and a variety of pathogenic proteoforms in plasma in a cohort of well-characterized VWD patients.

Methods: Citrated plasma samples from 10 healthy individuals and 65 participants of the Willebrand in the Netherlands (WiN) study were analyzed by (I) unbiased proteomics for VWF characterization and (II) targeted mass spectrometry for quantification. Proteins were digested with trypsin prior to desalting and LC-MS/MS analysis. To quantify VWF, VWFpp and five selected pathogenic VWF variants (C1190R, F1293L, R1306W, R1374H & Y1584C), stable-isotope labeled peptides were added as internal standard and results were calibrated to conventional units.

Results: Overall VWF sequence coverage was >70% and 16 pathogenic VWF proteoforms resulting from missense mutations, frame shifts or exon deletions were characterized. For the quantification of VWFpp and mature VWF, peptides in the D1 and C2 domains were selected based on peptide characteristics and inter-peptide agreements. VWF and VWFpp were quantified in all healthy individuals as well as in all type 1 and type 2 patients, but were not detected in VWD type 3. VWFpp/VWFmature ratios were increased in type 2 patients with a variant in the D'D3A1 domains. Surprisingly, we observed a spread in the variant-to-wild type stoichiometry, which was either < < 1 (C1190R and R1374H) or >> 1 (F1293L, Y1584C, R1306W).

Conclusion(s): Our study highlights the potential importance of circulating VWF proteoforms in plasma, which remains to be studied in relation to the VWD bleeding phenotype.