IDENTIFYING NOVEL HEMOSTATIC PROTEINS

动脉血栓形成与许多人类疾病有关，如急性心肌梗死和中风。很明显，大多数血栓事件需要血浆相凝血蛋白、循环中的血小板和局部血管内皮细胞之间的动态相互作用。关于液体相凝血蛋白和一些相关的血小板相互作用的调节已经有了详细的信息。尽管有这些信息，但很明显，在特定的血管床上调节动脉血栓形成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，我们在苏伦格博士和劳森博士的实验室之间制定了一个双层合作研究计划。第一层的目标是在相关的临床前模型中进行基于RNA的新型抗凝血剂和抗血栓化合物(适体)的翻译开发和评估，以测试针对血液凝血途径中不同猪促凝血剂因子的抑制剂的有效性和出血风险。第二层的目标是通过基于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，以分离这些以前未被鉴定的蛋白质的特定配体，并开始利用这些RNA配体在功能研究中揭开这些蛋白质的功能。这一合作研究项目的成功完成将需要苏伦格博士的实验室在基于RNA的组合化学方面和劳森博士的实验室在传统蛋白质生物化学、血液凝固分析和动脉血栓形成的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。分离与猪凝血因子Va、VIIa、VIIa、IXa、Xa和组织因子结合并抑制其功能的高亲和力核酸酶抗性RNA适配子，并在相关的临床前出血和抗凝模型中检测这些化合物。2.采用多靶点消减体外筛选技术，分离与这些未知蛋白特异性高亲和力结合的配体，鉴定血浆、血小板表面、病变血管内皮细胞和血管床特定血管节段中未知的血栓形成调节因子。3.评价铅RNA配体及其靶蛋白的单独或组合在生物测定中的抗血栓或促血栓作用，包括体外生化测定、血浆测定和活体猪出血、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.