Date of Award
Doctor of Philosophy In Clinical-bioanalytical Chemistry Degree
Yana Sandlers, Ph.D.
Bin Su, Ph.D.
Thrombomodulin (TM) is a transmembrane glycoprotein that is primarily expressed on the surface of endothelial cells, where it serves as a receptor of thrombin for protein C activation, which regulates coagulation and inflammation. Research has revealed that TM is also expressed in immune cells, including monocytes and macrophages, however, its function is unclear. In this dissertation study, the TM expression in THP-1 monocytes upon their differentiation to macrophages was profiled. Due to the beneficial roles of TM, the protein has caught the attention to be used as a therapeutic. A recombinant form of TM can be expressed that contains EGF-like domains 4-6 (rTM456) and this form, TM acts as pure anticoagulant while retaining the ability to activate protein C. However, this recombinant form has poor stability in vivo when left unmodified. Macrophages can be used as drug and antigen delivery carriers and can be directed to sites of inflammation. Modifying rTM456 to allow it to be incorporated into the membrane of macrophages may help with its stability and allow it to be deliver to sites of inflammation. One way to modify cells is lipid fusion by using lipid anchors that insert into the plasma membrane through hydrophobic interactions. In this dissertation, cell surface re-engineering of macrophages with rTM456 was investigated via a lipid fusion approach. First, the expression levels of TM were measured on THP-1 monocytes and macrophages. Using confocal microscopy and flow cytometry analysis, it was found that iv THP-1 monocytes express more TM on their cell surface compared to macrophages. It was also determined that THP-1 monocytes express more total TM, as determined by western blot and ELISA analysis. Western blot and ELISA data also revealed that monocytes shed significantly more TM into the culture medium when they are undergoing differentiation into macrophages versus resting monocytes. Despite expressing less TM on their cell surface, THP-1 macrophages were able to convert more protein C to activated protein C. Second, the anchoring efficiency of different lipid anchors for future cell surface re-engineering applications using a lipid fusion approach was investigated. Two different anchors were selected for this study, DSPE-PEG2000-Biotin and Cholesterol-PEG2000- Biotin. To determine anchoring efficiency, RAW 264.7 macrophages were incubated with different concentrations of DSPE-PEG2000-Biotin or Cholesterol-PEG2000-Biotin and tagged using streptavidin-FITC as a probe. Surface anchoring was determined using confocal microscopy. The cholesterol-based anchor showed drastically better incorporation efficiency than DSPE. In addition to incorporation efficiency, the membrane residence time of Cholesterol-PEG2000-Biotin was shown to have a concentration dependent loss of anchor from the cell surface with an overall retention half-life of 1hr. Last, a cell surface re-engineering strategy was developed for conjugating rTM456 to the surface of RAW 264.7 using a lipid fusion approach. Using a sortase A mediated ligation reaction, an azide was incorporated to the C-terminal end of the protein. This azide was used to attach a DBCO-terminated cholesterol anchor using copper free click chemistry. The anchoring efficiency of the afforded rTM-PEG2000-Cholesterol was then tested on RAW 264.7 macrophages. Confocal microscopy analysis showed that the rTM v conjugate could successfully anchor itself into the cell membrane. The insertion also causes little toxicity to the cell.
Boron, Mallorie L., "Expression And Cell Surface Re-engineering of Thrombomodulin on Macrophages" (2020). ETD Archive. 1248.