Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

Factor V Leiden (F5 ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5 (F5 ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi ). F8 deficiency enhanced the survival of F5Tfpi mice, demonstrating that F5Tfpi lethality is genetically suppressible. ENU-mutagenized F5 males and F5Tfpi females were crossed to generate 6,729 progeny, with 98 F5Tfpi offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3 ) suppressed F5Tfpi 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 F5Tfpi lethality (P = 1.7 × 10^-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 and also suggest a role for Actr2 in this process.