Date of Award

8-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Clinical-Bioanalytical Chemistry

Department

Chemistry

First Advisor

Sun, Xue-Long

Second Advisor

Zhou, Aimin

Third Advisor

Kothapalli, Chandrasekhar

Abstract

Thrombomodulin (TM), a membrane glycoprotein predominately expressed on endothelium, plays essential roles in regulating local hemostatic balance. Particularly, TM modulates the activity of thrombin from a procoagulant to an anticoagulant protease. When bound to TM, thrombin activates plasma protein C, which selectively inactivates coagulation factors Va and VIIIa. At the same time, TM-bound thrombin is unable to cleave fibrinogen or to activate platelets diminishing its procoagulant activity. recombinant TM456 is a promising anticoagulant candidate, but it has a very short half-life. TM is a proteoglycan and its O-linked glycosaminoglycan (GAG), chondroitin sulfate (CS), on the D3 domain contributes to TM’s additional thrombin binding activity as well as the protein’s stability, but production of proteoglycans/glycoproteins by recombinant techniques leads to uncontrolled glycoforms in desired proteins. In this dissertation, I expressed and purified rTM456 containing different bioorthogonal functional groups at the C-terminal using both sortase A mediated ligation and unnatural amino acid incorporation. Different CSs were synthesized with chain-end bioorthogonal functional groups so that they could be conjugated to the different rTM456 in a site-specific manner using bioorthogonal chemistry. In addition, azide and alkyne chain-end functionalized glycopolymers were synthesized for glyco-modification of rTM456 in a site-specific manner. The chain-end functional groups were confirmed using bioorthogonal reactions with fluorescent dyes. Lastly, glycopolymers and CS containing bioorthogonal functional groups were conjugated onto rTM456 at the C-terminal with the goal of developing an anticoagulant therapeutic, which is expected to have enhanced anticoagulant activity and biostability.

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