Cardiovascular disease (CVD) is the number one killer of mankind. Most CVDs are associated with atherosclerosis and thrombosis. It is well documented that platelets are the initiators of both atherosclerosis and thrombosis. Platelets are maintained in resting state in the circulation by endogenous negative regulators to avoid unintentional activation. During vascular injury, pro- stimulatory signals override anti-stimulatory signals to achieve rapid platelet activation. Lot of research is focused on pro-stimulatory signals while little is known about negative regulators. We have shown that JAM-A is an endogenous suppressor of platelet activation. Ablation of JAM-A confers an augmentation of in vivo thrombosis. However, upon platelet activation what happens to JAM-A is not known. We hypothesize that JAM-A forms a dimer upon dissociation from the integrin complex and support junctional assembly at the platelet–platelet junctions through the activation of Rap1. This R01 proposal is focused on delineating the role of JAM-A in initiating platelet-platelet junction formation, and thus preventing blood loss through the wound. Accordingly, three Specific Aims have been proposed. Specific Aim 1 will test the hypothesis that platelet JAM-A associates with the integrin through CD9, a tetraspanin, by forming a multi- protein complex in a PDZ-domain-dependent manner. We will use biochemical, mutational and genetic approaches to identify the components of this complex. Specific Aim 2 will test the hypothesis that upon platelet activation JAM-A dimerizes and assembles Rap1-activating complex to achieve Rap1 activation during outside-in signaling. We will use biochemical, mutational and genetic approaches to delineate the molecular mechanism that regulate JAM-A- dependent Rap1 activation. Specific Aim 3 will test the hypothesis that JAM-A is responsible in recruiting junctional proteins at the platelet-platelet contacts to assemble functional junctions. We will use genetically modified cells and mice to evaluate the role of JAM-A and other known junctional proteins in regulating permeability of hemostatic plug using in vitro and in vivo assays. Successful completion of this proposal will help to understand the role of junctional adhesion molecules in hemostasis and to develop therapeutic interventions for thrombosis associated diseases such as atherosclerosis, MI, and stroke.