End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. 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. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM₄₅₆ modification. Recombinant TM₄₅₆ with a C-terminal LPETG tag (rTM₄₅₆-LPETG) was expressed in Escherichia coli for its end-point modification with NH₂-diglycine-PEG₅₀₀₀-OMe via Sortase A-mediated ligation (SML). Similarly, 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 PEGylation of rTM₄₅₆via CFCC. Both PEGylated rTM₄₅₆ conjugates retained protein C activation activity as that of rTM₄₅₆. Also, they were more stable than rTM₄₅₆ in Trypsin digestion assay. Further, both PEGylated rTM₄₅₆ conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.