, the A3 domain of von Willebrand factor inducing combined qualitative and quantitative defects in the protein, Blood, vol.121, 2013.

I. Marx, P. J. Lenting, T. Adler, R. Pendu, O. D. Christophe et al., Correction of bleeding symptoms in von Willebrand factor-deficient mice by liver-expressed von Willebrand factor mutants, Arterioscler Thromb Vasc Biol, vol.28, pp.419-443, 2008.

C. Casari, V. Du, Y. P. Wu, A. Kauskot, P. G. De-groot et al., Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia, Blood, vol.122, 2013.

D. D. Wagner, S. Saffaripour, R. Bonfanti, J. E. Sadler, E. M. Cramer et al., Induction of specific storage organelles by von Willebrand factor propolypeptide, Cell, vol.64, pp.403-416, 1991.

H. Safdar, K. L. Cheung, D. Salvatori, H. H. Versteeg, H. Laghmani-el et al., Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production, Blood, vol.121, pp.4413-4419, 2013.

A. Eguchi, X. De-mollerat-du-jeu, C. D. Johnson, A. Nektaria, and A. E. Feldstein, Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis, J Hepatol, vol.64, pp.699-707, 2016.

E. Groot, R. Fijnheer, S. A. Sebastian, P. G. De-groot, and P. J. Lenting, The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission, J Thromb Haemost, vol.7, pp.962-971, 2009.

P. J. Lenting, E. Westein, V. Terraube, A. S. Ribba, E. G. Huizinga et al., An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation, J Biol Chem, vol.279, pp.12102-12111, 2004.

F. Bernardi, G. Marchetti, S. Guerra, A. Casonato, D. Gemmati et al., A de novo and heterozygous gene deletion causing a variant of von Willebrand disease, Blood, vol.75, pp.677-83, 1990.

F. Bernardi, P. Patracchini, D. Gemmati, M. Pinotti, C. Schwienbacher et al., In-frame deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease, Hum Mol Genet, vol.2, pp.545-553, 1993.

, Representative profile plots for multimers shown in B. Arrowheads indicate lighter heteropolymers. Dotted red lines highlight HMWMs visible after siDEL treatment, Chemi XT16 Image Systems and densitometric quantification was performed using

, As an estimation of the amount of the heteropolymers, the ratio between the intensity of the oddband and the intensity of the total band (WT-& odd-band intensities) was then calculated for each -MER. Three (siNEG) or four (siDEL) representative mice with sharp multimeric profiles before and after siRNA administration have been quantified. (E, upper panel) VWF antigen levels was measured at D3 & D5 in mice receiving siDEL (n=16, red), siNEG (n=4, gray) and in control untreated mice (n=19, black)

, Mice were divided between those who raised (grey) or lowered (white) their antigen levels at D5 compared to D3 post-HGT

, Statistical significance was calculated by applying 2-way ANOVA for repeated matched values with Sidak's multiple comparisons test using Prism 7 (Graphpad) software

. **-p<0, Error bars in the graphs indicate standard deviation from the mean

, Counteracting the dominant-negative effect of the heterozygous DEL-variant in vitro: DNA targeting approach

, COS-1 cells were transiently transfected with the same total amount of one (pSVhVWF/WT) or two plasmids (pSV-hVWF/WT & pSV-hVWF/DEL or pSV-hVWF/WT & pSV-hVWF/DEL/C2773R), in presence of Lipofectamine2000 (Life Technologies Corporation, Carlsbad, USA) essentially as previously described[12]. VWF multimeric composition and antigen levels were evaluated in cellular media

, ) as reference. Levels at D3 have been arbitrary set at 100% for this analysis. Statistical significance was calculated by applying a one-way ANOVA with Tukey's multiple comparisons using, COS-1 cells in high resolution 3% agarose gels, vol.7

, Counteracting the dominant-negative effect of the heterozygous DEL-variant in vivo: DNA targeting approach

, Vwf-deficient mice were subjected to HGT with one (control mice) or two plasmids (heterozygous VWD models). 4 days after HGT blood was collected and VWF assays performed on mouse plasma. (B) Representative multimer profile of plasmatic VWF in 2% agarose gels in mice co-expressing

. Vwf-wt and . Only, Arrowheads indicate heteropolymers containing DEL variants. (C) VWF antigen levels in mice expressing VWF-WT (n=21, black circles), both VWF-WT & VWF-DEL (n=52, red circles) and both VWF-WT & VWF

, VWF:Ag is expressed as % of a reference mouse pooled plasma from 10 female and 10 male C57Bl/6 mice. (D) A group of mice was subjected to tailclip bleeding assay 4 days after HGT. Blood loss in mice expressing VWF-WT, n=8, black circles; both VWF-WT & VWF-DEL, n=3, red circles; both VWF-WT & VWF-DEL/C2773R n=4, blue circles is shown. (E) Mice that were able to spontaneously arrest the bleeding in the tailclip assay have been referred as non-bleeders (white) and mice that never arrest the bleeding have been named bleeders (grey). The ratio of bleeders versus non-bleeders was similar in mice expressing mVWF-WT, DEL/C2773R (n=9, blue circles)

, Statistical significance was calculated by applying a one-way ANOVA with Tukey's multiple comparisons using Prism 7 (Graphpad) software

*. P<0, Error bars in the graphs indicate standard deviation from the mean