Thrombomodulin (TM), a membrane proteoglycan predominately expressed on endothelium, plays essential roles in keeping 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. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate, but has a very short half-life. TM is a proteoglycan and its O-linked glycosaminoglycan (GAG), chondroitin sulfate, 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. We report herein a recombinant rTM456 with a C-terminal LPETG motif for site-specific modification via sortase A-mediated ligation (SML). In addition, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific modification of rTM456 via copper-free click chemistry (CFCC). Both SML and CFCC facilitate glyco-engineering of rTM456 with defined GAG, which will enhance the rTM456’s anticoagulant activity and improve the stability of rTM456. The rTM456-chondroitin sulfate is expected to represent an effective and reliable anticoagulant protein drug for the treatment of thrombotic diseases.

Site-specific glyco-engineering of recombinant thrombomodulin

ACS Spring Meeting 2021

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