PB1436 PAR1 Knockout Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells Display Higher Tissue Factor-Dependent Thrombin Generation Compared to Wild Type

Research and Practice in Thrombosis and Haemostasis, vol. 7, pp. 101700

Abstract

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 nonselective 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.129Sinhibiting lung Vwf with 45% (B6-allele 9%, 129Sallele 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).

Authors 2

1. Wolfram Ruf Aachen Einthoven Laboratory for Vascular and Regenerative Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Internal Medicine , Division of Thrombosis and Hemostasis ,

   University Medicine of the

   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;

2. Leon J. Schurgers

   Maastricht University · Georgia Institute of Technology · Emory University · The Wallace H. Coulter Department of Biomedical Engineering

   Affiliation as printed

   Department of BIochemistry , Cardiovascular Research Institute Maastricht , Maastricht University , Maastricht , Limburg , Netherlands;

   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;

   Department of BIochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Limburg, Netherlands;

   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;