PROJECT SUMMARY Congenital deficiency of plasma clotting factor (F)XI is an autosomal disorder. Whereas some individuals are asymptomatic (non-bleeders), others have excessive bleeding after injury, primarily at sites with high fibrinolytic activity (mouth, nose, genitourinary tract) (bleeders). People with similarly reduced FXI have variable bleeding even within families sharing the same FXI mutation. Clinical assays cannot predict bleeding risk in FXI-deficient people, leading to under- or over-treatment to prevent bleeds. Moreover, FXI inhibition strategies are in clinical trials to reduce thrombosis; however, observations of individuals with congenital FXI deficiency suggest these therapies will incur bleeding risk in some patients, especially in prophylactic use. Uncovering mechanisms that determine bleeding risk in FXI deficiency and developing methods to predict bleeding will improve treatment for both bleeding and thrombosis. Our long-term goals are to characterize mechanisms that promote hemostasis in FXI deficiency, and translate these findings into clinically-accessible methods to predict bleeding. Using plasmas from two independent cohorts of well-phenotyped people with FXI deficiency, we developed and validated specialized plasma assays that differentiate FXI-deficient bleeders from non-bleeders, and discovered that inhibiting the contact pathway in these assays enhances the ability to identify bleeders. We also integrated computational modeling and in vitro assays to uncover synergy between FXIa and tissue factor that enhances coagulation. We built on these discoveries with new analyses that revealed plasma proteins whose levels differed significantly between non-bleeders and bleeders, and a novel computational workflow for advancing a prediction model. These findings and advances provide important clues to mechanisms that modify bleeding risk in FXI deficiency, and position us with innovative tools to identify these mechanisms. The objective of this application is to characterize the determinants and functional impact of differently present plasma proteins in non-bleeders and bleeders, and use computational methods to differentiate bleeding risk in FXI deficiency. The central hypothesis of this application is that in FXI deficiency, differences in plasma composition modify thrombin generation and clot formation, structure, and stability and determine the bleeding risk. Specific aims of this application are to: 1) Determine the functional impact and mechanisms differentiating differently present proteins in FXI-deficient non-bleeders and bleeders, 2) Use computational modeling and machine learning to identify predictive features that differentiate FXI-deficient non- bleeders and bleeders, and 3) Use multi-omic methods to define FXI deficiency and the bleeding phenotype. This proposed research is significant because the experiments will reveal molecular mechanisms that modify hemostatic potential in a predictive functional assay and ...