Thursday, November 21, 2024
11/21/2024
Project Summary Heparin is the most widely prescribed anticoagulant drug in the world and is used routinely for the treatment and prevention of deep vein thrombosis and pulmonary embolism. Currently, therapeutic heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa sourced from China. While essential and widely used, heparin has significant adverse complications. Approximately 600,000 patients per year treated with heparin in the USA develop a life-threatening condition known as heparin-induced thrombocytopenia (HIT), which results from the formation of heparin-platelet factor 4 (PF4) immunoreactive complexes. Therefore, there is an urgent need for safer, alternative sources of heparin. A recombinant source of heparin would be safer, avoid supply chain issues, and allow for the introduction of biological modifications to prevent HIT. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, there is a significant gap in knowledge regarding the regulatory mechanisms that give rise to the anticoagulant activity and biosynthesis of heparin. Heparin inhibits coagulation by binding with high affinity to the serine protease inhibitor, antithrombin (AT), and enhancing its activity to neutralize thrombin and Factor Xa in the coagulation cascade. Additionally, heparin exhibits high affinity binding to PF4, a chemokine produced by platelets, triggering HIT. Since PF4 binding to heparin depends on distinct binding sites compared to AT, we hypothesize that cells could be engineered to produce anticoagulant heparan sulfate with decreased PF4 affinity as a safer alternative to animal-derived heparin. Our previous studies have revealed distinct regulatory mechanisms for heparin biosynthesis in cells, suggesting that other factors exist that regulate anticoagulant heparin/HS production and modify AT and PF4 affinity. The goal of this proposal is to leverage our experience in genome-wide screening assays to identify genetic factors that control heparin biosynthesis and can be utilized for bioengineering anticoagulant heparan sulfate in cultured cells. To accomplish this goal, we aim to (i) adapt genome-wide screening assays to search for novel factors that regulate AT and PF4 binding to cell surface heparan sulfate, and (ii) validate prioritized hits from the screens and leverage these for metabolic engineering of mammalian cells to produce anticoagulant heparan sulfate with lowered PF4 affinity. The successful completion of these aims will provide a bioengineered cell line that produces a safer, recombinant form of heparin and may also uncover previously unknown genes associated with heparin’s activity and assembly. Importantly, this project will lead to future detailed hypothesis-driven studies that will bring us closer to finding an alternative to animal-derived heparin for improving patient care and clinical outcomes by prevention of HIT.
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