Thrombotic diseases such as myocardial infarction, stroke, and thromboembolism are severe with significant mortality and morbidity in the United States. Antithrombotic agents can be used for both prevention and treatment of active vascular thrombosis. However, current antithrombotic therapy is limited by the risk of bleeding, hemorrhagic complication. Recent advances in molecular bases of haemostasis have highlighted new targets for novel antithrombotic agent design. Endothelial thrombomodulin (TM) plays a critical role in local haemostasis by binding thrombin and subsequently converting protein C to its active form (APC), which is an anticoagulant protease that selectivelyinactivates coagulation factors VA and VIIIa. In addition, the binding of thrombin to TM drastically alters the thrombin's procoagulant activities to anticoagulant activities. Importantly, TM expression, however, decreases in perturbed endothelial cells, predisposing to thrombotic occlusion and particularly in response to a variety of inflammatory stimuli, direct vessel wall injury, and oxidant stress. TM is a type I membrane protein. 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. Liposomes, in which lipid composition closely resembles that of cell membranes, have been extensively studied as cell membrane model as well as carrier for delivering certain vaccines, enzymes, drugs, or genes to their active sites. Therefore, we propose a TM-liposome conjugate to mimic the native endothelial antithrombotic mechanism of both TM and lipid components and thus will provide a more forceful than current antithrombotic agent. Using membrane protein as a drug presents special challenges since it is difficult to purify and manipulate an amphiphilic membrane protein and difficult to maintain the active form of a membrane protein as in cell membrane. In this proposal, we want to test a central hypothesis that recombinant and chemo- and bio-orthogonal membrane-mimetic assembling membrane protein thrombomodulin (recombinant TM-liposome conjugate) provides a potent antithrombotic agent and a rational design strategy for generating a membrane mimetic drug.
The Specific Aims are the following: (1)Synthesize and characterize recombinant TM (rTM)- liposome conjugates in chemo-/bio-orthogonal approach;(2) Evaluate in vitro antithrombotic activity of the rTM-liposome conjugates;(3) Define the capacity of rTM-liposome conjugates to limit coagulation events as well as their pharmacokinetics in vivo.
Thrombotic disorders continue to represent a major cause of morbidity and mortality in the United States despite available methods of diagnosis and treatment. Currently available anticoagulants share the common property of disrupting normal hemostatic pathways. Anticoagulation is often accompanied by hemorrhagic or other side effects, which necessitate interruption of therapy. Furthermore, no beneficial effects in preventing restenosis after revascularization procedures have yet been obtained with the established antithrombotic agents. Thus, an antithrombotic agent that is safer and more effective than currently available is highly demanded. Recent understanding of haemostasis in the molecular bases has highlighted new targets for novel antithrombotic agent design. Physiologically, endothelial thrombomodulin (TM) plays a critical role in local haemostasis. However, TM expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion and particularly in response to a variety of inflammatory stimuli, direct vessel wall injury, and oxidant stress. In this proposal, we want to develop a recombinant and chemo-/bio-orthogonal approach to synthesize liposomal TM conjugate that mimics the native endothelial antithrombotic mechanism of both TM and lipid components and thus would be a novel and more potent antithrombotic agent.
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|Weingart, Jacob; Vabbilisetty, Pratima; Sun, Xue-Long (2013) Membrane mimetic surface functionalization of nanoparticles: methods and applications. Adv Colloid Interface Sci 197-198:68-84|
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