OC 17.2 - Type 2N VWD VWF variants and a more common VWF variant show defects in VWF-FVIII binding in a mammalian cell-surface display system

Sunday, June 23, 2024

09:45 – 10:00 ICT

Room: 210 A-D

Presenting Author(s)

Jill Johnsen, MD

Associate Professor
Department of Medicine, University of Washington, Seattle, WA, US; Institute for Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA US
Seattle, Washington, United States

Co-Author(s)

SF

Shelley Fletcher

Scientist
Bloodworks Northwest
Seattle, Washington, United States
NP

Nicholas Popp

MSTP Student
University of Washington
Seattle, Washington, United States
Pamela Christopherson

Senior Research Coordinator
Blood Research Institute, Versiti BloodCenter of Wisconsin, Milwaukee, WI, US.
Milwaukee, Wisconsin, United States
DH

Daniel Hampshire

Lecturer in Genetics
Biomedical Institute for Multimorbidity, Centre for Biomedicine, Hull York Medical School, Hull, UK.
Hull, England, United Kingdom
AY

Andrew Yee

Assistant Professor
Baylor College of Medicine
Houston, Texas, United States
DF

Doug Fowler

Professor
University of Washington
Seattle, Washington, United States
KL

Kerry Lannert

Research Scientist
University of Washington
Seattle, Washington, United States

Background: von Willebrand factor (VWF) is a large plasma protein critical in hemostasis. VWF protects factor VIII (FVIII) in circulation. Type 2N von Willebrand disease (VWD) is characterized by impaired VWF-FVIII binding. It is critical to distinguish type 2N VWD from low FVIII due to hemophilia A. VWF genotyping can inform accurate diagnoses. A growing number of DNA variants are being identified in the VWF gene. However, the interpretation of pathogenicity is often hampered by insufficient evidence.

Aims: We adapted our multiplexed cell surface tethering of extracellular proteins (MultiSTEP) system to display VWF and test the functional impact of VWF variants on VWF-FVIII binding.

Methods: We constructed a VWF cDNA with the regions encoding the VWF propeptide deleted (∆Pro-VWF). ∆Pro-VWF was cloned into our MultiSTEP cassette that provides a C-terminal flexible linker and transmembrane domain. We studied five VWF missense variants – four found in type 2N VWD (c.2446 C>T;p.R816W), c.2561 G>A;p.R854Q, c.2572 T>A;p.C858S, c.2635 G>A;p.D879N) and one common variant c.2451 T>A;p.H817Q. Variants were subcloned and recombined into the genomically integrated MultiSTEP landing pad in HEK293 Freestyle cells. VWF-displaying cells were incubated with recombinant FVIII, stained with anti-FVIII or anti-VWF antibodies, then fluorescently-conjugated secondary antibodies. Cell surface VWF and FVIII abundance were determined by flow cytometry.

Results: Cell-surface displayed ∆Pro-VWF robustly displays in our MultiSTEP system and binds FVIII. All VWF missense variants express VWF at normal levels. All variants show impaired VWF-FVIII binding (Figure 1).

Conclusion(s): We have developed an in vitro mammalian cell surface display system for the interrogation of VWF variant impact on VWF-FVIII binding. Our data provide strong in vitro evidence for pathogenicity in type 2N VWD and give useful information regarding the potential of p.H817Q to contribute to a type 2N VWD-like phenotype. These in vitro data enabled us to re-classify the pathogenicity of VWF variants with confidence in VWD.