PROJECT SUMMARY Platelets are a blood component critical to hemostasis, i.e. the prevention of blood loss at sites of vascular injury. For hemostasis, platelets must activate their primary integrin (aIIbb3) through the small GTPases Rap1 and downstream protein Talin. Integrin activation is essential to platelet adhesion and aggregation. Activated platelets can also release their granule contents and undergo phospholipid scrambling to support the formation of a stable hemostatic plug. The mechanisms of platelet activation must be tightly controlled as hypoactive platelets cause prolonged bleeding while hyperactive platelets are associated with thrombotic disease. Platelets which undergo phospholipid scrambling expose a negatively charged phospholipid, phosphatidylserine (PtdSer), to their outer membrane surface. These so called procoagulant platelets recruit and activate coagulation factors resulting in the generation of thrombin and the formation of fibrin, an integral component of stable hemostatic plugs. Loss of PtdSer exposure or coagulation factors results in bleeding complications; conversely, excessive PtdSer exposure is associated with increased thrombotic risk. Despite the negative outcomes associated with dysregulation of PtdSer, there exists a gap in knowledge on the mechanisms regulating PtdSer exposure following platelet activation. The small GTPase Rap1 is a well-established regulator of platelet integrin activation and aggregation. Loss of Rap1 in vitro also results in decreased PtdSer exposure; however, the mechanism and in vivo significance of Rap1-dependent PtdSer exposure are unknown. Interestingly, Rho family GTPases (RhoA/Rac/Cdc42) also modulate PtdSer exposure in platelets, and studies in multiple cell types, including platelets, have demonstrated crosstalk between Rap1 and Rho GTPases. Therefore, I hypothesize that Rap1-dependent PtdSer exposure occurs through crosstalk with Rho family small GTPases, and that Rap1-dependent procoagulant activity is critical during hemostatic plug formation. Preliminary data supports my hypothesis as inhibition of RhoA signaling leads to increased PtdSer exposure in platelets lacking both Rap1 isoforms, Rap1A and Rap1B. I have also established a novel imaging platform which allows for the study of platelet-dependent procoagulant response during hemostasis in vivo. In aim 1 I will characterize the crosstalk between Rap1 and Rho Family GTPases during platelet activation leading to PtdSer exposure. In aim 2 I will use my novel 4-D imaging model of hemostasis to characterize how Rap1 signaling affects platelet- coagulation interplay in vivo. This work will expand our understanding of the regulation of Rap1 mediated platelet PtdSer exposure and its consequences in vivo.