Significance of the amino acid at position 2561 in the C4 domain of Von Willebrand factor
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Background
The von Willebrand factor (VWF) Phe2561Tyr variant has been previously shown to exhibit gain-of-function–like activity and increase the risk of repeated myocardial infarction in patients aged <55 years. It was hypothesized that altered stem dynamics enhanced the responsiveness of the molecule to shear stress.
Objectives
In this study, we investigated the evolutionary significance of the amino acid at position 2561 and functional impacts of variants at this site.
Methods
Variants at 2561 identified by protein sequence alignments were generated by mutagenesis and expressed in HEK293T cells and function investigated using static and flow-based assays and biophysical methods.
Results
Phe2561 was heavily conserved in mammals; however, nonmammals occupied the site with tyrosine, threonine, or serine. Recombinant expression of Phe2561Tyr/Thr/Ser showed no effects on expression, multimers, collagen or glycoprotein IIbIIIa binding, but Ser2561 and Thr2561 demonstrated enhanced binding to gain-of-function glycoprotein Ibα. While none of the variants, including Tyr2561, enhanced VWF-mediated platelet capture to collagen at 1500/s or 5000/s, the Tyr2561, Ser2561, and Thr2561 variants exhibited enhanced formation of rolling VWF-platelet aggregates, with Ser2561 demonstrating the greatest gain-of-function activity. Optical tweezer pulling experiments using VWF-D'CK dimers revealed altered unfolding properties of the Tyr, Thr, and Ser2561 protein and atomic force microscopy imaging of VWF dimeric stems demonstrated that Tyr, Thr, and Ser2561 favored a more open stem state, suggesting this is responsible for the enhanced response to shear stress.
Conclusion
This study indicates that the amino acid at 2561 is crucial for modulating VWF function by helping to mediate stem dynamics.
The von Willebrand factor (VWF) Phe2561Tyr variant has been previously shown to exhibit gain-of-function–like activity and increase the risk of repeated myocardial infarction in patients aged <55 years. It was hypothesized that altered stem dynamics enhanced the responsiveness of the molecule to shear stress.
Objectives
In this study, we investigated the evolutionary significance of the amino acid at position 2561 and functional impacts of variants at this site.
Methods
Variants at 2561 identified by protein sequence alignments were generated by mutagenesis and expressed in HEK293T cells and function investigated using static and flow-based assays and biophysical methods.
Results
Phe2561 was heavily conserved in mammals; however, nonmammals occupied the site with tyrosine, threonine, or serine. Recombinant expression of Phe2561Tyr/Thr/Ser showed no effects on expression, multimers, collagen or glycoprotein IIbIIIa binding, but Ser2561 and Thr2561 demonstrated enhanced binding to gain-of-function glycoprotein Ibα. While none of the variants, including Tyr2561, enhanced VWF-mediated platelet capture to collagen at 1500/s or 5000/s, the Tyr2561, Ser2561, and Thr2561 variants exhibited enhanced formation of rolling VWF-platelet aggregates, with Ser2561 demonstrating the greatest gain-of-function activity. Optical tweezer pulling experiments using VWF-D'CK dimers revealed altered unfolding properties of the Tyr, Thr, and Ser2561 protein and atomic force microscopy imaging of VWF dimeric stems demonstrated that Tyr, Thr, and Ser2561 favored a more open stem state, suggesting this is responsible for the enhanced response to shear stress.
Conclusion
This study indicates that the amino acid at 2561 is crucial for modulating VWF function by helping to mediate stem dynamics.
Date Issued
2025-12-01
Date Acceptance
2025-08-19
Citation
Journal of Thrombosis and Haemostasis, 2025, 23 (12), pp.3858-3870
ISSN
1538-7933
Publisher
Elsevier BV
Start Page
3858
End Page
3870
Journal / Book Title
Journal of Thrombosis and Haemostasis
Volume
23
Issue
12
Copyright Statement
© 2025 The Author(s). Published by Elsevier Inc. on behalf of International Society on Thrombosis and Haemostasis. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/40915573
PII: S1538-7836(25)00548-3
Subjects
Shear-stress
Thrombosis
Von Willebrand Factor
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2025-09-05