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

脂质体血栓调节蛋白的重组和化学/生物正交合成

• 批准号: 8223139
• 负责人: XUE-LONG SUN
• 金额: $ 30.8万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-09 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223139
• 关键词: Active Sites; Adverse effects; Anticoagulants; Anticoagulation; Azides; Binding; Blood Coagulation Factor; Blood Vessels; C-terminal; Cell membrane; Coagulation Process; Complication; Diagnosis; Disease; Drug Kinetics; Endothelial Cells; Enzymes; Event; Factor Va; Fibrinolytic Agents; Genes; Hemorrhage; Hemostatic Agents; Hemostatic function; In Vitro; Inflammatory; Injury; Interruption; Ligation; Lipid Bilayers; Lipids; Liposomes; Membrane; Membrane Proteins; Methods; Molecular; Morbidity - disease rate; Myocardial Infarction; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Play; Prevention; Procedures; Property; Protein C; Reaction; Recombinants; Risk; Role; Site; Stimulus; Stroke; Testing; Thrombin; Thromboembolism; Thrombomodulin; Thrombosis; United States; Vaccines; active method; base; cofactor; design; in vivo; membrane model; mimetics; mortality; mouse model; novel; oxidant stress; prevent; public health relevance; response; restenosis

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.

描述（由申请人提供）：在美国，心肌梗塞、中风和血栓栓塞等血栓性疾病非常严重，死亡率和发病率很高。抗血栓药物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、出血并发症的风险的限制。止血分子基础的最新进展突出了新型抗血栓剂设计的新目标。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)，在局部止血中发挥关键作用，APC 是一种抗凝蛋白酶，可选择性地灭活凝血因子 VA 和 VIIIa。此外，凝血酶与TM的结合极大地将凝血酶的促凝血活性转变为抗凝血活性。然而，重要的是，TM 表达在受到干扰的内皮细胞中减少，容易发生血栓闭塞，特别是在对各种炎症刺激、直接血管壁损伤和氧化应激的反应中。 TM是I型膜蛋白。它所在的脂质双层充当重要的“辅助因子”，局部集中并协调反应辅助因子和蛋白 C 激活底物的适当排列。脂质体的脂质成分与细胞膜的成分非常相似，已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进行了广泛的研究。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，从而提供比现有抗血栓剂更有效的抗血栓剂。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维持膜蛋白如在细胞膜中的活性形式。在这个提案中，我们想要测试一个中心假设，即重组和化学和生物正交的膜模拟组装膜蛋白血栓调节蛋白（重组TM-脂质体缀合物）提供了一种有效的抗血栓剂和产生膜模拟药物的合理设计策略。具体目标如下：（1）采用化学/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活性； (3) 定义rTM-脂质体缀合物限制凝血事件的能力及其体内药代动力学。 公共卫生相关性：尽管有可用的诊断和治疗方法，血栓性疾病仍然是美国发病和死亡的主要原因。目前可用的抗凝剂具有破坏正常止血途径的共同特性。抗凝治疗通常伴有出血或其他副作用，需要中断治疗。此外，现有的抗血栓药物尚未获得预防血运重建手术后再狭窄的有益效果。因此，迫切需要一种比目前可用的更安全、更有效的抗血栓剂。最近对分子基础止血的理解突出了新型抗血栓剂设计的新目标。从生理学角度来说，内皮血栓调节蛋白（TM）在局部止血中发挥着关键作用。然而，TM 表达在受到干扰的内皮细胞中减少，容易发生血栓闭塞，特别是在对各种炎症刺激、直接血管壁损伤和氧化应激的反应中。在本提案中，我们希望开发一种重组和化学/生物正交方法来合成脂质体TM缀合物，该缀合物模拟TM和脂质成分的天然内皮抗血栓机制，因此将成为一种新型且更有效的抗血栓剂。