IDENTIFYING NOVEL HEMOSTATIC PROTEINS

鉴定新型止血蛋白

• 批准号: 6527459
• 负责人: BRUCE ALAN SULLENGER
• 金额: $ 26.95万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527459
• 关键词: RNA; anticoagulants; blood coagulation; blood proteins; coagulation factor IX; coagulation factor V; coagulation factor VII; coagulation factor VIII; coagulation factor X; combinatorial chemistry; inhibitor /antagonist; ligands; oligonucleotides; protein structure function; thrombosis

Arterial thrombosis is associated with a large array of human pathology such as acute myocardial infarction and stroke. It is clear that most thrombotic events require a dynamic interplay between plasma phase blood coagulation proteins, circulating platelets, and local vascular endothelial cells. Detailed information has been developed regarding the regulation of fluid phase blood coagulation proteins and some associated platelet interactions. In spite of this information, it is clear that the regulation of arterial thrombosis in specific vascular beds requires a more detailed understanding of vascular specific interactions with plasma phase proteins and cellular elements. To this end we have developed a two-tiered collaborative research program between the laboratories of Dr. Sullenger and Dr. Lawson. The goal of the first tier is the translational development and evaluation in relevant pre-clinical models of novel RNA-based anticoagulant and antithrombotic compounds (aptamers) to test the efficacy versus bleeding risk associated with inhibitors that target different porcine procoagulant factors in the blood coagulation pathway. The goal of the second tier is the identification of novel plasma phase, platelet and vascular bed specific proteins and their genes by using RNA- based combinatorial chemistry methods to isolate specific ligands to these previously uncharacterized proteins, and to start to unravel the function of these proteins by employing these RNA ligands in functional studies. The successful completion of this collaborative research program will require the joint expertise and personnel of Dr. Sullenger's laboratory in RNA-based combinatorial chemistry and of Dr. Lawson's laboratory in conventional protein biochemistry, blood coagulation assays and animal models of arterial thrombosis. Thus, our specific aims are: l. To isolate high affinity nuclease-resistant RNA aptamers that bind to and inhibit the function of porcine clotting factors Va, VIIa, VIIIa, IXa, Xa and tissue factor, and test these compounds in relevant pre-clinical models of bleeding and anticoagulation. 2. To identify yet unknown regulators of thrombosis in the plasma, on the surface of platelets and on endothelial cells of diseased and vascular bed specific vessel segments by employing multi-target subtractive in vitro selection techniques to isolate ligands capable of specific and high affinity binding to these previously uncharacterized proteins. 3. To assess the anti- or pro-thrombotic effects of individual or combinations of lead RNA ligands and their target proteins in bioassays, including in vitro biochemical assays, plasma based assays and in vivo swine models of bleeding, anticoagulation and arterial injury.

动脉血栓形成与大量人类病理学相关，例如急性心肌梗死和中风。很明显，大多数血栓形成事件需要血浆相凝血蛋白、循环血小板和局部血管内皮细胞之间的动态相互作用。 关于流体相凝血蛋白的调节和一些相关的血小板相互作用的详细信息已经被开发。尽管有这些信息，但很明显，在特定血管床中动脉血栓形成的调节需要更详细地了解与血浆相蛋白和细胞成分的血管特异性相互作用。为此，我们在Sullivan博士和Lawson博士的实验室之间开发了一个两级合作研究计划。第一层的目标是在相关临床前模型中对新型基于RNA的抗凝剂和抗血栓化合物（适体）进行转化开发和评价，以测试与靶向凝血途径中不同猪促凝血因子的抑制剂相关的疗效与出血风险。第二层的目标是通过使用基于RNA的组合化学方法来鉴定新的血浆相、血小板和血管床特异性蛋白质及其基因，以分离这些先前未表征的蛋白质的特异性配体，并通过在功能研究中使用这些RNA配体来开始阐明这些蛋白质的功能。 这项合作研究计划的成功完成将需要Sullivan博士实验室在基于RNA的组合化学和Lawson博士实验室在常规蛋白质生物化学，血液凝固测定和动脉血栓形成动物模型方面的联合专业知识和人员。因此，我们的具体目标是：l。分离高亲和力核酸酶抗性RNA适体，其结合并抑制猪凝血因子Va、VIIa、VIIIa、IXa、Xa和组织因子的功能，并在出血和抗凝的相关临床前模型中测试这些化合物。2.通过采用多靶点消减体外选择技术分离能够特异性和高亲和力结合这些先前未表征的蛋白质的配体，鉴定血浆、血小板表面以及病变和血管床特异性血管段内皮细胞中血栓形成的未知调节因子。3.在生物测定（包括体外生化测定、基于血浆的测定和出血、抗凝和动脉损伤的体内猪模型）中评估前导RNA配体及其靶蛋白的单独或组合的抗血栓形成或促血栓形成作用。