Browsing by Subject "Plasmin"

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    Multivalent Benzamidine Molecules for Plasmin Inhibition: Effect of Valency and Linker Length
    (Wiley, 2022) Nallan Chakravarthula, Tanmaye; Zeng, Ziqian; Alves, Nathan J.; Emergency Medicine, School of Medicine
    There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis‐associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono‐, bi‐ and tri‐valent) and linker lengths (∼1–12 nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S‐2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri‐AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7 μM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length.
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    The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity
    (Springer, 2024) Esser, Nathalie; Hogan, Meghan F.; Templin, Andrew T.; Akter, Rehana; Fountaine, Brendy S.; Castillo, Joseph J.; El‑Osta, Assam; Manathunga, Lakshan; Zhyvoloup, Alexander; Raleigh, Daniel P.; Zraika, Sakeneh; Hull, Rebecca L.; Kahn, Steven E.; Medicine, School of Medicine
    Aims/hypothesis: Apart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer's disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes. Methods: The expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes. Results: In isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1-11 and 12-37 fragments. hIAPP 12-37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05). Conclusions/interpretation: The fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.