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

Factor V Leiden (F5L ) 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 F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/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 F5L/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 F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.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 Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

Homozygosity for factor V Leiden leads to enhanced thrombosis and atherosclerosis in mice.

BACKGROUND: Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial. METHODS AND RESULTS: To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type (Fv+/+), heterozygous FVL (FvQ/+), and homozygous FVL (FvQ/Q) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. FvQ/Q mice formed occlusive thromboses 27+/-3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv+/+ mice (56+/-7 minutes, n=9, P<0.01), whereas FvQ/+ mice (41+/-7 minutes, n=5) were intermediate (P=0.5, compared with Fv+/+). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv+/+ and FvQ/Q mice. FvQ/Q mice transplanted with Fv+/+ bone marrow formed occlusive thromboses at 35+/-5 minutes (n=7, P<0.05 compared with Fv+/+ mice), whereas Fv+/+ mice transplanted with FvQ/Q bone marrow occluded at 59+/-7 minutes (n=6, P<0.001 compared with FvQ/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, FvQ/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)-deficient strain (ApoE-/-) to generate FvQ/Q,ApoE-/- mice. By 52 weeks of age, FvQ/Q,ApoE-/- mice (n=8) had developed more aortic atherosclerosis (40+/-6% lesion area) compared with Fv+/+,ApoE-/- mice (15+/-3% lesion area; n=12, P<0.02). CONCLUSIONS: In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non-platelet-derived plasma FVL.