Recombinant and Chemo-/Bio-Orthogonal Synthesis of Liposomal Thrombomodulin

描述（由申请人提供）：血栓性疾病，如心肌梗死、中风和血栓栓塞是严重的，在美国具有显著的死亡率和发病率。抗血栓药物可用于预防和治疗活动性血管血栓。然而，目前的抗血栓治疗受到出血和出血性并发症风险的限制。止血分子基础的最新进展为新型抗血栓药物的设计提供了新的靶点。内皮凝血调节蛋白（TM）通过结合凝血酶并随后将蛋白C转化为其活性形式（APC），在局部止血中起关键作用，APC是一种抗凝蛋白酶，可选择性地激活凝血因子VA和viia。此外，凝血酶与TM的结合使凝血酶的促凝活性急剧改变为抗凝活性。然而，重要的是，TM表达在受干扰的内皮细胞中降低，易于血栓闭塞，特别是在各种炎症刺激、直接血管壁损伤和氧化应激的反应中。TM是I型膜蛋白。它所在的脂质双分子层作为一种必需的“辅助因子”，局部集中和协调反应辅助因子和底物的适当排列，以激活蛋白C。脂质体的脂质组成与细胞膜的脂质组成非常相似，作为细胞膜模型以及将某些疫苗、酶、药物或基因运送到其活性位点的载体，脂质体已被广泛研究。因此，我们提出了一种TM-脂质体缀合物来模拟TM和脂质成分的天然内皮抗血栓机制，从而提供比目前的抗血栓剂更有效的抗血栓药物。利用膜蛋白作为药物具有特殊的挑战，因为难以纯化和操作两亲性膜蛋白，并且难以维持膜蛋白在细胞膜中的活性形式。在这个提议中，我们想要验证一个中心假设，即重组和化学和生物正交的模拟膜组装膜蛋白血栓调节蛋白（重组tm -脂质体偶联物）提供了一种有效的抗血栓剂，并为生成模拟膜药物提供了合理的设计策略。具体目的如下：(1)利用化学/生物正交法合成重组TM (rTM)-脂质体偶联物并进行表征；(2)评价rtm -脂质体偶联物的体外抗血栓活性；(3)确定rtm -脂质体偶联物限制凝血事件的能力及其体内药代动力学。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.