Date of Award

2012

Degree Type

Dissertation

Department

Chemistry

First Advisor

Sun, Xue-Long

Subject Headings

Thrombomodulin, Liposomes, Antithrombins, Chemistry, Analytic, Liposomal antithrombotic thrombomodulin conjugation

Abstract

Recent advances in the molecular bases of haemostasis have highlighted that endothelial thrombomodulin (TM) plays a critical role in local haemostasis by binding thrombin and subsequently converting protein C to its active form (APC). In addition, the binding of thrombin to TM drastically alters the thrombin's procoagulant activities to anticoagulant activities. The lipid bilayer in which it resides serves as an essential 'cofactor', locally concentrating and coordinating the appropriate alignment of reacting cofactors and substrates for protein C activation. On the other hand, liposomes have been extensively studied as cell membrane model as well as carrier for delivering certain vaccines, enzymes, drugs, or genes. In this study, antithrombotic liposomal TM conjugate was design and developed by combination of antithrombotic membrane protein TM into liposome through recombinant and bioorthogonal conjugation techniques, which providing a rational strategy for generating novel and potential antithrombotic agent. Namely, the liposomal TM conjugate mimics the native endothelial antithrombotic mechanism of both TM and lipid components and thus is more forceful than current antithrombotic agent.First, an efficient and chemoselective liposome surface functionalization method was developed based on Staudinger ligation, in which a model compound carbohydrate derivative carrying a spacer with azide was conjugated onto the surface of preformed liposomes carrying a terminal triphenylosphine in PBS buffer (pH 7.4) and at room temperature.Second, recombinant TM containing the EGF-like domains 456 with an azidohomoalaine at the C-terminal has been expressed and incorporated into liposome to form antithrombotic liposomal TM conjugate via Staudinger ligation. In addition, another chemically selective liposomal surface functionalization method for antithrombotic liposomal TM conjugated has been developed based on copper-free click chemistry, which provides an alternative approach to improving reaction efficiency and stability and

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