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

描述(由申请人提供)：血栓性疾病，如心肌梗塞、中风和血栓栓塞症，在美国非常严重，死亡率和发病率都很高。抗血栓药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、出血性并发症风险的限制。近年来，止血分子基础的研究进展为新型抗血栓药物的设计提供了新的靶点。内皮血栓调节蛋白(TM)通过与凝血酶结合，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是一种抗凝血酶，可选择性地抑制凝血因子VA和VIIIa。此外，凝血酶与TM的结合极大地改变了凝血酶的促凝血活性和抗凝活性。然而，重要的是，TM在扰乱的内皮细胞中的表达减少，容易发生血栓闭塞，特别是对各种炎症刺激、直接血管壁损伤和氧化应激的反应。TM是一种I型膜蛋白。它所在的脂质双层是一种基本的辅助因子，局部集中并协调反应辅助因子和底物的适当排列，以激活蛋白C。脂质体具有与细胞膜相似的脂类成分，作为细胞膜模型以及将某些疫苗、酶、药物或基因输送到其活性部位的载体，已被广泛研究。因此，我们提出了一种TM-脂质体结合物来模拟TM和脂质成分的天然内皮抗血栓机制，从而提供了一种比目前更有效的抗血栓药物。膜蛋白作为药物具有特殊的挑战性，因为两亲性膜蛋白很难纯化和操作，而且很难保持膜蛋白在细胞膜中的活性形式。在这项提议中，我们想要检验一个中心假设，即重组和化学和生物正交膜模拟组装膜蛋白血栓调节蛋白(重组TM-脂质体结合物)为产生膜模拟药物提供了一种有效的抗血栓药物和合理的设计策略。具体目标如下：(1)用化学/生物正交法合成和表征重组TM(RTM)-脂质体结合物；(2)评价RTM-脂质体结合物的体外抗血栓活性；(3)确定RTM-脂质体结合物限制凝血事件的能力及其在体内的药代动力学。 公共卫生相关性：血栓性疾病仍然是美国发病率和死亡率的主要原因，尽管有可用的诊断和治疗方法。目前可用的抗凝剂具有扰乱正常止血途径的共同特性。抗凝常常伴随出血或其他副作用，这就需要中断治疗。此外，现有的抗血栓药物在预防血管重建术后再狭窄方面尚未取得任何有益的效果。因此，迫切需要一种比目前可用的更安全和更有效的抗血栓药物。最近对分子基础止血的了解为新型抗血栓药物的设计提供了新的靶点。从生理上讲，内皮细胞血栓调节蛋白(TM)在局部止血中起着关键作用。然而，在扰动的内皮细胞中，TM的表达减少，易于发生血栓闭塞，特别是对各种炎症刺激、直接血管壁损伤和氧化应激的反应。在这项建议中，我们希望开发一种重组和化学/生物正交法来合成脂质体TM结合物，它模仿TM和脂质成分的天然内皮抗血栓机制，从而成为一种新的、更有效的抗血栓药物。

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.