OC 37.1 - Advanced hemostasis testing by quantitative proteomics

Monday, June 24, 2024

09:30 – 09:45 ICT

Room: 210 A-D

Presenting Author(s)

T. T. van Duijl, T.T.

Post-doctoral researcher
Molecular Hematology, Sanquin Research, Asterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands

Co-Author(s)

AK

Alette Kooiker

Master student
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
IK

Iris C. Kreft

PhD candidate
Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
Amaury Monard

PhD candidate
Internal Medicine, Maastricht University Medical Center +, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
Caroline Mussert, MD

PhD candidate
Department of hematology – pediatric hematology, Erasmus Medical Center Sophia Children's Hospital, University Medical Center Rotterdam, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
Cv

Carmen van der Zwaan

Senior technician
Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
JM

Joost C.M. Meijers

Professor
Department of Experimental Vascular Medicine, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.
Amsterdam, Noord-Holland, Netherlands
Yvonne Henskens

Professor of the department of clinical chemistry
MUMC+
maastricht, Limburg, Netherlands
MC

Marjon H. Cnossen

MD, PhD, professor
Department of Pediatric Hematology and Oncology, Erasmus MC Sophia Children’s Hospital, University Medical Center Rotterdam, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
MK

Marieke J.H.A. Kruip

Hematologist
Department of Hematology, Erasmus MC, Erasmus University Medical Centre Rotterdam, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
Floor Heubel-Moenen

MD, PhD
Department of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands
Frank W.G Leebeek, MD PhD

Internist-hematologist, Professor
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Mv

Maartje van den biggelaar

Principal investigator
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands

Background: Embedding of proteomics in hemostasis screening may overcome excessive sequential antigen testing. Mass spectrometry-based proteomic enables quantification of protein sequence variants unique to pro-or anticoagulant proteoforms contributing to hemostasis.

Aims: Molecular footprinting of bleeding disorders by quantitative proteomics with sensitivity towards procoagulant proteoforms.

Methods: Proteomics was applied to quantify proteins involved in coagulation and fibrinolysis, primarily based on the Tier 1 diagnostic gene list endorsed at the ISTH2023. Stable isotope-labelled peptides were added as internal standards and a calibration strategy was developed for quantification in conventional units. External quality controls and protein-specific deficient plasma’s (n=18) were used for analytical performance monitoring. Proteomic signatures were obtained in bleeding disorders, including Von Willebrand Disease (VWD)(n=64), dysfibrinogenemia (n=3), FV proteoform-directed bleeding (n=3) and bleeding disorder of unknown cause (BDUC)(n=24).

Results: The proteomic strategy enabled quantification of VWF(pp), ADAMTS13, PF4, PPBP, fibrinogen (α,β,γ chain), FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIIIB and regulators AT, HCII, PROC, PROS, PROZ, KIN-1, A2AP, PLG, TAFI and TFPI from 10 μL plasma, but PAI-1, FXIIIA and FVIII were typically below the analytical limit of quantification (≈ 5 nM). Differential coagulation protein-deficient plasmas mimicked partial (25%; 50%; 75%) and complete deficiencies. Determination of the VWFpp/VWF ratio in patients with various types of VWD highlighted VWF proteoforms of increased clearance (Figure1). Sequence variants from pathogenic proteoforms of VWF and fibrinogen γ were directly quantified from plasma (Figure2), whereas the FV-short proteoforms were recognized indirectly by enhanced TFPI. Hemostatic signatures were normal in most BDUC patients, with few exceptions (e.g. HCII) from which the pathogenicity of abnormal levels remain to be evaluated.

Conclusion(s): The proteomic screening approach enabled identification of common and rare bleeding disorders and was piloted for diagnosis of exclusion in BDUC. Collaboration initiatives are initiated to dissect the biological etiologies contributing to hemostatic imbalance and address the unmet diagnostic needs.