ABSTRACT Refrigerated storage reduces platelet life-span because it causes cytoskeletal rearrangements, de- sialylated glycoprotein-Ib (GPIb) to cluster, form microdomains, shed and induces mitochondrial dependent reactive oxygen species (ROS) and apoptosis, which may result in inflammatory response in vulnerable patient populations. Recognition by host of clustered glycoproteins (GP) results in platelet phagocytosis and clearance. As a consequence, cold stored platelets are only allowed for use in trauma patient therapy and not for prophylaxis or treatment of stem cell transplant recipients and hematology/oncology patients. The Rho family GTPases RHOA and RAC1 are central regulators of cytoskeletal rearrangements, and have been shown to control lipid raft formation and composition; changes in Rho GTPase activities may influence platelet membrane lipid raft assembly, post- translational modifications of membrane glycoproteins, included GpIb and increased mitochondrial ROS and apoptotic activity. Our preliminary, submitted and published data using genetic and pharmacological means show that reversible RHOA GTPase inhibition results in an inhibition of myosin activity and prevention of clathrin-independent formation and internalization of lipid rafts enriched in active glycosyl-transferases (GT) and GPIb. RHOA GTPase inhibition prevents the metabolic reprogramming effect and allows the maintenance of glycolytic flux and mitochondrial dependent respiration and ROS production. Importantly, we further demonstrate that murine, human and Rhesus- macaque platelets, when stored in refrigerated conditions for up to 14 days in the presence of a lead RHOA inhibitor, G04, can retain survival function at a level similar to that of room-temperature stored platelets and retain hemostatic activity in vivo, and an antioxidant phenocopies some of the effects of G04. We hypothesize that RHOA controls the process of GP clustering during cold storage through the regulation of actomyosin activity, vesicle trafficking and mitochondrial respiration. We will first identify the mechanism by which RHOA regulates lipid raft formation, GP clustering and endocytosis in platelets upon refrigeration. We will also determine the outcomes of pharmacologic inhibition of RHOA in preventing the metabolic and mitochondrial damage of long-term cold stored platelets by analyzing the effect of long-term storage on mitochondrial activity and the crosstalk between RHOA and the master metabolic regulator AMPK in regulating platelet metabolism and mitophagy. Our studies will provide the mechanism and a stringent proof-of-principle for the translational value of a novel approach to refrigerated platelet storage.