Thursday, April 3, 2025
4/3/2025
Plasminogen and Plasmin: Structure and Function - Project Summary Mature human plasminogen (Glu1-hPg) is a 791-amino acid residue single-chain protein that is the inactive precursor of the two-chain disulfide-linked serine protease, plasmin (hPm). The conversion of hPg to hPm occurs by proteolytic cleavage of a single peptide bond catalyzed by hPg activators (PAs). One highly characterized function of hPm is lysis of the fibrin clot. However, the overall functions of hPm are much more complex. hPm formed in plasma is rapidly inactivated by the plasma inhibitor a2- antiplasmin (a2AP), resulting in an inactive a2AP/hPm complex. Thus, unless very large amounts of hPm are formed, e.g., during thrombolytic therapy, hPm would have a very limited function in solution. Therefore, the overall hypothesis of this proposal is that hPm bound to specific receptors (Pg-Rs) on cells is protected from a2AP and provides these cells with a proteolytic surface, which can be utilized for physiological purposes, e.g., control of hemostasis, and pathological events, e.g., migration, dissemination, and invasion. Levels of circulating hPm are also limited by the native T-conformation of hPg, which is highly resistant to activation. Thus, we propose to determine the molecular nature of the intramolecular interactions of hPg that stabilize its T-conformation and the molecular forces responsible for binding of hPg to Pg-Rs through in vitro, ex vivo, in vivo, and in situ experiments. Three specific aims are presented in this multi-PI proposal to address these hypotheses. Specific aim 1 proposes to generate hPg variants to examine the binding forces that stabilize the T-conformation of hPg and the forces that govern its binding to mammalian and bacterial Pg-Rs. Hypothesis: By genetically designing proteins and cells with altered hPg/hPm and receptor conformation and binding, we will gain a deeper understanding of the binding and functions of Pg-R/hPg-hPm interactions. Specific Aim 2 will study the in vivo effects on inflammation and hemostasis of genetically-altered mice and bacterial receptors. Hypothesis: using the knowledge gained in Aim 1, we will rationally design mice and bacteria with altered components of the Pg-R pathways and identify the mechanisms of the different stages of virulence. Specific aim 3 presents studies to investigate, in situ, how cell and tissue microenvironments influence the host response to bacterial infection as a consequence of hPg/Pg-R interactions by building and expanding innovative 2-D and 3-D cellular model systems. Hypothesis: SK- mediated activation of hPg bound to the surface of bacteria is a major driver of invasive outcomes, which can be precisely modeled using physiologically-detailed models of tissue and cell-based systems. Impact: The major impact of this work is the development of a comprehensive roadmap of host and virulence determinants related to Pg-Pg-R interactions involved in bacterial infection using multiple approaches from a variety of disciplines, thus allowing design of potential new classes of drugs.
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