Project Summary The long-term goal of our work is to identify genetic variants that suppress intravascular blood clotting called thrombosis or venous thromboembolism (VTE). We previously use a genome wide mutation screen in mice to identify 10 dominant genetic variants that suppressed lethal thrombosis. Several of these mutations are in genes which, when mutated, enable mice with a lethal thrombosis genotype to survive. Arl6ip5 is one such gene. This gene and its protein product, also known as JWA, Praf3 etc. has multiple putative cellular and organismal functions, overlapping with Actr2 (Arp2, part of the Arp2/3 complex). Our central hypothesis is that Arl6ip5 deficiency affects thrombosis by altering essential cellular functions via cytoskeletal and transcriptional pathways. These in turn affect platelet reactivity, gene transcription, and the kinetics of thrombosis. We will complete the Specific Aims of our project by performing assays on our genome edited mouse model of Arl6ip5 deficiency. Specific AIM 1 will investigate the ability of complete Arl6ip5 deficiency to increase thrombosis suppression relative to partial Arl6ip5 deficiency. We will also investigate the effects of Arl6ip5 on tail bleeding and Rose Bengal induced arterial and venous thrombosis. Specific AIM 2 experiments will probe Arl6ip5 deficient platelets for functional and transcriptional defects using targeted platelet assays and platelet RNA sequencing. To follow up on the plasma activated partial thrombosplastin time (APTT) defect we observed in Arl6ip5 deficient mice, we will perform detailed studies on blood and plasma, including the measurement of coagulation factor levels and liver RNA sequencing to determine the molecular genetic pathway leading to this effect. Both male and female mice will be testing to ensure the rigor and thoroughness of our studies. The completion of the proposed studies will provide the first evidence of the role Arl6ip5 gene mutations play in regulating hemostasis. These questions are highly impactful to the mission of the NIH because they provide critical information about the regulation of thrombi, a widespread killer causing heart attacks, strokes, and VTE. Increased fundamental knowledge of this process could provide better insights into preventative and therapeutic strategies. These studies will also be used to train the next generation of cardiovascular researchers in the proper conduct of scientific studies, preparing them for outstanding independent scientific careers in cardiovascular research. The funding of this grant will further contribute to the outstanding training environment of the Oakland University Department of Biological Sciences, which has been doing an outstanding job of contributing scientists to graduate and postdoctoral training programs in cardiovascular research.