Recombinant and chemo-/bio-orthogonal synthesis of liposomal thrombomodulin and its antithrombotic activity

Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. The EGF-like domains 4-6 of TM (TM₄₅₆) are essential for PC activation. In this study, we proposed a liposomal recombinant TM conjugate to mimic the membrane TM structure and its anticoagulant activity. First, a DSPE-PEG₂₀₀₀-TM₄₅₆ was successfully synthesized by site-specific conjugation of azido-TM₄₅₆ with DSPE-PEG₂₀₀₀-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM₄₅₆ was fabricated via direct liposome formation with the DSPE-PEG₂₀₀₀-TM₄₅₆ and other lipids. This liposomal formulation of TM₄₅₆ retained protein C activation activity as that of TM₄₅₆. Also, liposome-TM₄₅₆ was much more stable and had a longer plasma half-life than TM₄₅₆ and DSPE-PEG₂₀₀₀-TM₄₅₆, respectively. Moreover, liposome-TM₄₅₆ showed in vivo anticoagulant effect by decreasing the mortality from 80% to 20% in a thrombin-induced thromboembolism mouse model. The reported liposome-TM₄₅₆ conjugate mimics the endothelial TM anticoagulation activity and may serve as an effective anticoagulant agent candidate for future development.