%0 Journal Article %A Randal J. Westrick %A Kärt Tomberg %A Amy E. Siebert %A Guojing Zhu %A Mary E. Winn %A Sarah L. Dobies %A Sara L. Manning %A Marisa A. Brake %A Audrey Cleuren %A Lena M. Mishack %A Linzi M. Hobbs %A Alexander Johnston %A David R. Siemieniak %A Jishu Xu %A Jun Z. Li %A David Ginsburg %T A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci %D 2016 %R 10.1101/080432 %J bioRxiv %P 080432 %X Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism (VTE), though it exhibits only 10% penetrance. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−). The observation that F8 deficiency enhanced survival of F5L/L Tfpi+/− mice demonstrated the potential for genetic suppression of F5L/L Tfpi+/− lethality. G0 ENU-mutagenized F5L/L males and F5L/+ Tfpi+/− females were next crossed to generate 6,739 G1 progeny, with 98 F5L/L Tfpi+/− offspring surviving until weaning and 16 exhibiting transmission of a putative thrombosuppressor to subsequent generations. The resulting lines are referred to as MF5L, (Modifier of Factor 5 Leiden 1-16). Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-induced F3 mutation was identified, heterozygous F3 deficient mice (F3+/-) suppressed F5L/L Tfpi+/− lethality. Thus, like F8 deficiency, reduced F3 activity suppresses F5L/L Tfpi+/− thrombosis. Whole exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Attempts to generate an independent Actr2 knockin/knockout via CRISPR/Cas9 failed for any mouse beyond the 60-cell stage. Our findings identify F8 and the TFPI/F3 axis as key regulators of thrombosis balance in the setting of F5L and demonstrate the utility of this sensitized ENU mutagenesis approach for the identification of dominant thrombosis suppressor loci. %U https://www.biorxiv.org/content/biorxiv/early/2016/10/16/080432.full.pdf