Cardiovascular disease (CVD) is the number one killer of mankind. Most CVDs are associated with atherosclerosis and thrombosis. Mounting evidence suggests that platelets are the initiators of both atherosclerosis and thrombosis. Agonist stimulation in platelets is known to activate MAPKs, and it has been shown that they are important for platelet activities both in vivo and in vitro. Despite this evident role of MAPK signaling contributing to platelet functions, the mechanisms through which they regulate platelet activities are not fully understood. We have identified that a member of the MAP3K family, apoptosis signal-regulating kinase (ASK1) is present in both human and murine platelets and is activated by physiological agonists. We have shown that Ask1 activity supports platelet aggregation and secretion, and ablation of Ask1 confers a protective effect in in vivo models of thrombosis. We therefore hypothesize that platelet ASK1 is a key regulator of atherogenesis, atherothrombosis, and ischemia reperfusion (I/R) injury resulting from clot dissolution in MI, and stroke. We have also identified several structurally distinct ASK1 inhibitors based on information from published virtual chemical library screens. Two of these compounds have shown excellent efficacy in protecting mice from thrombosis with minimal effects on hemostasis, as assessed by tail bleeding time and laser-induced hemostasis model. This R01 proposal is focused on delineating the role of platelet ASK1 in initiating atherogenesis, atherothrombosis, and aggravating I/R injury. Accordingly, three Specific Aims have been proposed. Specific Aim 1 will test the hypothesis that platelet ASK1 is key in initiating atherogenesis. We will use a hyperlipidemia mouse model (Apoe-/- mice fed with high-fat diet) to study the effect of ablation or inhibition of platelet Ask1 on plaque formation. Specific Aim 2 will test the hypothesis that platelet ASK1 is a central regulator of platelet activation during atherosclerotic plaque rupture (atherothrombosis). We will use an innovative mouse model to mimic thrombus formation at the site of plaque rupture. Specific Aim 3 will test the hypothesis that platelet ASK1 is responsible for aggravating I/R injury. We will use the transient middle cerebral artery occlusion model (tMCAO) of stroke to assess the effect of ablation/inhibition of platelet ASK1 on I/R injury. Successful completion of this proposal will help to develop a number of therapeutic interventions for thrombosis associated diseases such as atherosclerosis, MI, and stroke.