IDENTIFYING NOVEL HEMOSTATIC PROTEINS

动脉血栓形成与许多人类病理有关，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期凝血蛋白、循环血小板和局部血管内皮细胞之间的动态相互作用。关于液相凝血蛋白的调控和一些相关的血小板相互作用的详细信息已经得到了发展。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血管与血浆相蛋白和细胞元件的特定相互作用。为此，我们在Sullenger博士和Lawson博士的实验室之间制定了一个两层合作研究计划。第一阶段的目标是在相关的临床前模型中对新型基于rna的抗凝血和抗血栓化合物（适配体）进行转化开发和评估，以测试针对血液凝固途径中不同的猪促凝因子的抑制剂的疗效和出血风险。第二层的目标是鉴定新的血浆期、血小板和血管床特异性蛋白质及其基因，通过基于RNA的组合化学方法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配体开始揭示这些蛋白质的功能。这项合作研究计划的成功完成将需要Sullenger博士的rna组合化学实验室和Lawson博士的传统蛋白质生物化学、血液凝固测定和动脉血栓动物模型实验室的联合专业知识和人员。因此，我们的具体目标是：1 .分离高亲和力的核酸酶抗性RNA适配体，结合并抑制猪凝血因子Va、VIIa、VIIa、IXa、Xa和组织因子的功能，并在相关的出血和抗凝血临床前模型中对这些化合物进行测试。2. 通过采用多靶点减法体外选择技术分离能够与这些先前未表征的蛋白质特异性和高亲和力结合的配体，确定血浆、血小板表面和病变血管床特定血管段内皮细胞中尚不清楚的血栓形成调节因子。3. 评估单个或组合铅RNA配体及其靶蛋白在生物分析中的抗或促血栓作用，包括体外生化分析、血浆分析和猪体内出血、抗凝和动脉损伤模型。

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.