PROJECT SUMMARY Our research program centers on the regulation of hemostasis and thrombosis, heavily informed by the study of human patients with associated genetic disorders. The current proposal will continue this work, with a specific focus in four areas: 1) characterization of endothelial cell heterogeneity across diverse vascular beds and its role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome, and other vascular disorders, 2) application of comprehensive deep mutational scanning methods, using von Willebrand factor (VWF) and plasminogen activator inhibitor-1 (PAI-1) as model hemostasis proteins, to guide precision medicine genome sequence interpretation, 3) directed molecular evolution of novel serine protease inhibitor (SERPIN) proteins with unique new properties, providing valuable insight into protein structure/function relationships as well as identifying potential novel therapeutics, and 4) characterization of the full cargo repertoires for the LMAN1 and SURF4 cargo receptors, identifying potential overlapping cargo receptors, and probing unconventional protein secretion, with important implications for disorders of hemostasis and lipid metabolism. We will identify and characterize a novel modifier gene for TTP susceptibility in the mouse, with potential relevance for the variable presentation of TTP in humans. We will also characterize endothelial gene expression across diverse vascular beds in vivo at the single cell level in mouse models for TTP and other vascular disorders. Building on our past work, we will assemble complete datasets encompassing every possible single amino acid substitution within PAI-1 and select domains of VWF to markedly improve the diagnosis of human disorders due to inherited mutations in these genes and interpretation of clinical genome sequencing data. These data will also provide a powerful resource for exploring structure–function relationships within these proteins. We will also apply these approaches to probe the determinants of target protease specificity for PAI-1 and related members of the SERPIN gene family, with the potential to enable the development of novel therapeutic reagents for the treatment of hemostatic and other human diseases. Finally, we will continue our studies of the ER to Golgi cargo receptors, LMAN1/MCFD2, required for efficient secretion of coagulation factors V and VIII, and SURF4, the cargo receptor for PCSK9 and other proteins regulating plasma cholesterol, to identify the full repertoire of cargo for each receptor, as well as potential overlapping cargo receptors, with important implications for patients with associated genetic disorders, as well as the development of future novel therapeutics.