PROJECT SUMMARY / ABSTRACT The long-term goal of this proposal is to advance transfusion effectiveness and safety by addressing the gaps in knowledge of testosterone-mediated cellular changes that impact red blood cell (RBC) and platelet function in storage and after transfusion. We hypothesize that disruption of physiological testosterone signaling induced by genetic, idiopathic or therapeutic intervention (testosterone replacement therapy, TRT) contributes to inter-donor heterogeneity in the quality of blood components, and to altered transfusion effectiveness. The scientific premise is based on our findings that supraphysiological concertation (>890 ng/dL; common in TRT) of free (bioavailable) testosterone in plasma is correlated with increased oxidative hemolysis and decreased membrane deformability. Relevant to transfusion effectiveness, gamma-irradiated RBC units from TRT donors had lower survival shortly after infusion into immunodeficient mice compared with non-TRT controls, and patients’ hemoglobin increments following a single RBC unit transfusion were reduced for RBC units from TRT donors compared to those from non-TRT male donors (75% of non-TRT). Our studies in platelets suggested that exogenous testosterone has a priming effect on platelets evidenced by increased aggregation and mitochondrial bioenergetics, and that TRT was associated with increased expression of platelet activation markers in storage. In Aim 1, we will quantify the concentrations of free and total testosterone in plasma from male donors from NHLBI’s REDS-III RBC-Omics study with complete genotyped information and linked data of recipient outcomes after RBC transfusion events. By measuring plasma testosterone concentration, we will be able to determine the prevalence of sub- physiological (hypogonadism) and supraphysiological testosterone among male donors; conduct genome-wide association (GWA) studies to identify single nucleotide polymorphism (SNP) associated with donor plasma testosterone concentration; and define the impact of testosterone concentration and identified SNPs on transfusion effectiveness (measured by changes in patient’s hemoglobin and bilirubin increments) using the REDS-III vein-to-vein database. In Aim 2, we evaluate the impact of hypogonadism and TRT on platelet phenotype and hemostatic function in therapy, in storage, and in a mouse model of transfusion. The goal is to evaluate the feasibility of expanding the utilization of blood components derived from individuals on TRT. We anticipate that this project’s outcomes will allow us to: identify genetic determinants of plasma testosterone that impact the effectiveness of testosterone therapy in patients, and RBC/platelet survival in storage and after transfusion; benefit the emerging field of precision transfusion medicine by establishing blood donor testosterone threshold values for transfusion effectiveness and safety in vulnerable patient populations (neonates/androgen sensitive); improve TRT ...