Abstract Venous thromboembolism (VTE) susceptibility genes are largely unknown. We used a sensitized ENU mutagenesis screen of 6,739 mice to identify novel dominant thrombosis suppressor genes of the perinatal lethal F5 Leiden homozygous (F5L/L), tissue factor pathway inhibitor (Tfpi+/-) heterozygous phenotype. These highly penetrant, dominant mutations restored viability to the F5L/L Tfpi+/- mice and were able to be successfully passed on to offspring. We named these suppressor genes MF5L for Modifier of Factor 5 Leiden. In order to identify the MF5L13 suppressor, we used whole exome sequencing to directly identify a single candidate suppressor mutation in exon 7 of the Actr2 gene. This arginine (R) to glycine (G) mutation is in the highly conserved amino acid position 258 of the ARP2 protein (Actr2+/G) and suppresses thrombosis by an as yet unknown mechanism. The studies proposed here seek to functionally characterize the MF5L13 suppressor mutation. Preliminary experimental results on platelet aggregation and platelet dependent thrombus formation of Actr2+/G compared to wildtype Actr2+/+ mice have suggested a platelet functional defect in the Actr2+/G mutants. Comparative analysis of liver Serpine2 mRNA levels via qPCR also revealed a dramatic increase in Serpine2 levels in Actr2+/G compared to Actr2+/+ mice. In the proposed studies, we will first use CRISPR/Cas9 genome editing to re- create the Actr2+/G in a clean (non-mutagenized) mouse genetic background. Using this coisogenic Actr2+/G mouse, we will first test its ability to thrombosuppress F5L/L Tfpi+/-. This mouse model will then be crossed to the Tfpi deficient background (Tfpi-/-) to genetically dissect the thrombosuppressive mechanisms of Actr2+/G relative to F5L/L and Tfpi. To determine whether there are more global expression changes associated with Actr2+/G or if Serpine2 alone is affected, we will perform platelet and liver RNAseq. We will then conduct a series of experiments designed to determine the thrombosuppressive mechanism(s) of the Actr2+/G. We will determine the effects of Actr2+/G on platelet actin filament assembly rates, the effects on agonist induced platelet aggregation and platelet activation events, phosphatidylserine exposure, and thrombin generation, F5 activity and in vivo arterial and venous thrombosis induction. This work will lead to new insights into the mechanisms of action of a unique thrombosuppressor gene and may lead to new therapeutic targets for human thrombotic disease.