Factor V Leiden (F5^L ) 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^L (F5^L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi^+/- ). F8 deficiency enhanced the survival of F5^L/LTfpi^+/- mice, demonstrating that F5^L/LTfpi^+/- 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/LTfpi^+/- 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 F5^L/LTfpi^+/- 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/LTfpi^+/- 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^L and also suggest a role for Actr2 in this process.

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