A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

Factor V Leiden ( F5 L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5 L ( F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor ( Tfpi +/− ). F8 deficiency enhanced survival of F5 L/L Tfpi +/− mice, demonstrating that F5 L/L Tfpi +/− lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/− females were crossed to generate 6,729 progeny, with 98 F5 L/L Tfpi +/− offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to as MF5L ( M odifier of F actor 5 L eiden) 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, haploinsufficiency for F3 ( F3 +/− ) suppressed F5 L/L Tfpi +/− lethality. Whole exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5 L/L Tfpi +/− lethality (p=1.7x10 −6 ), suggesting that Actr2 p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action for Actr2 p.R258G . Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a novel role for Actr2 in this process. Significance Statement Venous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.