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 ( Fv Q /+ ), and homozygous FVL ( Fv Q/Q ) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. Fv Q/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 Fv Q /+ 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 Fv Q/Q mice. Fv Q/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 Fv Q/Q bone marrow occluded at 59±7 minutes (n=6, P <0.001 compared with Fv Q/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, Fv Q/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)–deficient strain ( ApoE −/− ) to generate Fv Q/Q ,ApoE −/− mice. By 52 weeks of age, Fv Q/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.

Factor V Leiden (F5 L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-Nnitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for) and haploinsufficient for tissue factor pathway inhibitor (Tfpi ), 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.venous thromboembolism | Factor V Leiden | ENU mutagenesis | tissue factor pathway inhibitor | genetic screen

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 (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, 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 (120 words max):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.

Factor V Leiden, (FVL) is the most common known inherited thrombotic risk factor and is present in approximately 5% of most Western populations and 25–50% of patients presenting with venous thrombosis. However, FVL is incompletely penetrant, with only approximately 10% of FVL carriers developing thrombosis in their lifetimes. Though interactions between FVL and other known prothrombotic mutations have been documented in a few cases, the genetic factors responsible for the incomplete penetrance of FVL remain largely unknown. We previously reported a remarkable synthetic lethality in mice carrying the FVL mutation and partial deficiency of a key coagulation component, tissue factor pathway inhibitor (TFPI). Complete TFPI deficiency in mice is embryonic lethal, whereas heterozygosity is compatible with normal survival. However, homozygosity for FVL (FvQ/Q) in the context of heterozygosity for TFPI (Tfpi+/−) is uniformly lethal due to disseminated perinatal thrombosis. In order to identify potential modifier genes contributing to FVL penetrance, we have utilized this lethal genetic interaction as a phenotyping tool for a sensitized ENU mutagenesis screen in laboratory mice. We hypothesize that dominant mutations in key components of the coagulation system will improve hemostatic balance and allow survival in mice carrying the lethal FvQ/Q Tfpi+/− genotype combination. As an example, we propose that loss of one tissue factor allele might compensate for reduced TFPI and rescue FvQ/Q Tfpi+/− . To test this hypothesis, we bred tissue factor heterozygous mice (Tf+/−) with FvQ/Q Tfpi+/− mice and observed complete rescue, with normal survival and the expected number (8 of 57) of FvQ/Q Tfpi+/− Tf+/− mice from a FvQ/+ Tfpi+/− Tf+/−x FvQ/Q cross. In order to identify candidate modifier genes, we performed a whole genome mutagenesis screen. In this screen, male FvQ/Q mice were mutagenized with ENU and bred to FvQ/+ Tfpi+/− double heterozygous females. DNAs from surviving offspring were PCR assayed to identify rescued mice with the FvQ/Q Tfpi+/− genotype. Analysis of 2250 offspring, corresponding to approximately half genome coverage, has identified 15 mice that survived to weaning. Heritability was demonstrated for the 5 mutant lines subjected to progeny testing to date. Genetic crosses are in progress to map the mutant genes in 3 of the 5 progeny tested lines. These preliminary results demonstrate the feasibility of this sensitized screen for the identification of dominant suppressors of thrombosis. Based on our data, we estimate that there are likely 10–20 mammalian genes for which a <50% reduction in expression could result in a major shift in hemostatic balance sufficient to rescue the lethal thrombosis associated with the FvQ/Q Tfpi+/− lethal genotype. Each of these loci represent a candidate for a human modifier gene in patients with FVL and other thrombophilic mutations. Finally, the biologic pathways uncovered by these studies should provide new insights into the overall regulation of hemostatic balance and identify potential new targets for therapeutic intervention.