PB1431 - Effect of allele-selective silencing of von Willebrand factor in mice on experimental bleeding and thrombosis models

Tuesday, June 27, 2023

18:30 – 19:30 ET

Room: Exhibition

Co-Author(s)

Yvonne K. Jongejan, MSc

PhD candidate
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
Leiden, Zuid-Holland, Netherlands

Presenting Author(s)

Noa A. Linthorst, MSc

PhD candidate
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
Leiden, Zuid-Holland, Netherlands

Co-Author(s)

ES

Elisa Schrader

PhD candidate
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, Georgia, United States.
Atlanta, Georgia, United States
Sebastiaan N.J Laan, MSc

PhD candidate
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
RD

Richard J. Dirven

Research technician
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
leiden, Zuid-Holland, Netherlands
JD

James E. Dahlman

Associate professor
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, Georgia, United States.
Atlanta, Georgia, United States
Bv

Bart J.M. van Vlijmen

Associate professor
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
Leiden, Zuid-Holland, Netherlands
Cécile V. Denis, PhD

Principal investigator, Director of Research
Laboratory for Hemostasis, Inflammation and Thrombosis, Unité Mixte de Recherche S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
Le Kremlin-Bicêtre, Ile-de-France, France
Jeroen C.J Eikenboom, MD. PhD

Professor, Head of Division of Thrombosis and Hemostasis
Leiden University Medical Center
Leiden, Zuid-Holland, Netherlands

Background: Von Willebrand factor (VWF) plays an important role in the initiation of platelet adhesion. Defective VWF causes bleeding and high VWF plasma levels are associated with thrombosis. We have shown inhibition of endothelial murine Vwf using allele-selective small interfering RNAs (siRNAs) targeting either C57BL/6J (B6) or 129S1/SvImJ (129S) mice.

Aims: To investigate the effect of allele-selective Vwf-silencing in F1 crosses of B6 and 129S mice on bleeding and thrombosis.

Methods: B6.129S F1 hybrid mice were intravenously injected with nanoparticle-encapsulated siRNAs. 96 hours post-injection, citrated blood and lungs were collected for measurement of plasma VWF:Ag and lung Vwf mRNA levels. Allele-selectivity was tested for siRNAs targeting either Vwf expressed from B6- (siVwf.B6) or 129S-alleles (siVwf.129S), using a non-selective siVwf and scrambled siControl as controls. siVwf.B6-injected B6 and B6.129S mice were subjected to a ferric chloride (FeCl3) experimental thrombosis model (mesenteric vessels) and a tail-clip bleeding assay.

Results: Both siRNAs were allele-selective, with siVwf.B6 inhibiting lung Vwf with 41% (B6-allele 72%, 129S-allele 12%) and plasma VWF with 46% (Figure 1), and siVwf.129S inhibiting lung Vwf with 45% (B6-allele 9%, 129S-allele 58%) and plasma VWF with 43%. siVwf.B6-mediated allele-selective Vwf-silencing in B6.129S mice lead to a minor increase (+24 seconds) in bleeding time compared to siControl.B6-treated B6.129S mice, whereas siVwf.B6-treated B6 mice showed a moderate to severe increase (+120 seconds) in bleeding time (Figure 2A). FeCl3-treatment only rarely lead to thrombotic occlusions in B6.129S mice. However, in siVwf.B6-treated B6 mice only 50% of the mice developed an occlusion versus 89% of the siControl.B6-treated mice (Figure 2B).

Conclusion(s): Allele-selective siRNAs effectively and selectively inhibit Vwf at mRNA and plasma level. 50% VWF reduction had no relevant impact on the bleeding time. The effect of 50% VWF reduction on the FeCl3-thrombosis model was not conclusive and is being analyzed.
Funded by the Dutch Thrombosis Foundation (grant #2018-01).