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、卒中和血栓栓塞在美国是严重的，具有显著的死亡率和发病率。抗血栓剂可用于预防和治疗活动性血管血栓形成。然而，目前的抗血栓治疗受到出血风险、出血性并发症的限制。止血分子基础的最新进展突出了新型抗血栓药物设计的新靶点。内皮血栓调节蛋白（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是一种选择性激活凝血因子VA和VIIIa的抗凝蛋白酶。此外，凝血酶与TM的结合将凝血酶的促凝血活性急剧改变为抗凝活性。然而，重要的是，TM表达在扰动的内皮细胞中减少，易患血栓性闭塞，特别是响应于各种炎症刺激、直接血管壁损伤和氧化应激。TM是I型膜蛋白。它所在的脂质双层作为一个重要的“辅因子”，局部集中和协调反应辅因子和蛋白C激活底物的适当对齐。脂质体的脂质组成与细胞膜的脂质组成非常相似，已被广泛研究作为细胞膜模型以及用于将某些疫苗、酶、药物或基因递送至其活性位点的载体。因此，我们提出了一种TM-脂质体结合物，以模拟TM和脂质成分的天然内皮抗血栓形成机制，从而提供比目前更有力的抗血栓形成剂。使用膜蛋白作为药物提出了特殊的挑战，因为难以纯化和操作两亲性膜蛋白，并且难以保持膜蛋白的活性形式如在细胞膜中。在这个提议中，我们想测试一个中心假设，即重组和化学和生物正交的膜模拟组装膜蛋白血栓调节素（重组TM-脂质体缀合物）提供了一个有效的抗血栓形成剂和一个合理的设计策略，产生一个膜模拟药物。具体目的如下：（1）以化学/生物正交方法合成和表征重组TM（rTM）-脂质体缀合物;（2）评价rTM-脂质体缀合物的体外抗血栓形成活性;（3）确定rTM-脂质体缀合物限制凝血事件的能力以及它们在体内的药代动力学。

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.