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 (TM(456)) 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-PEG(2000)-TM(456) was successfully synthesized by site-specific conjugation of azido-TM(456) with DSPE-PEG(2000)-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM(456) was fabricated via direct liposome formation with the DSPE-PEG(2000)-TM(456) and other lipids. This liposomal formulation of TM(456) retained protein C activation activity as that of TM(456). Also, liposome-TM(456) was much more stable and had a longer plasma half-life than TM(456) and DSPE-PEG(2000)-TM(456), respectively. Moreover, liposome-TM(456) showed in vivo anticoagulant effect by decreasing the mortality from 80% to 20% in a thrombin-induced thromboembolism mouse model. The reported liposome-TM(456) conjugate mimics the endothelial TM anticoagulation activity and may serve as an effective anticoagulant agent candidate for future development.