Novel Technologies for Imaging Blood Coagulation Reactions

描述(由申请人提供)：我们建议解决挑战(06-HL-103)，提供使能技术，旨在开发新的定量成像方法，用于测量完整小鼠的凝血酶功能，以提高对细胞相互作用的基本了解，凝血的生物途径及其在流动血液中的调节。活体荧光显微镜检查激光诱导的小鼠提睾主动脉损伤后血栓形成的方法为定量研究活体生物血液中的血栓形成开辟了新的途径。然而，该方法依赖于使用荧光抗体的间接措施来选择反应物。它的全部前景受到了试剂和方法的限制，这些试剂和方法可以直接测量凝血的各个步骤。我们建议利用我们在凝血反应生物化学方面的成熟专业知识，结合一系列具有明确特性的凝血蛋白部位特异性荧光衍生物，开发针对凝血酶复合体组装和功能的灵敏和定量测量的使能技术。我们提出的方法旨在研究凝血酶定位和血栓演变之间的空间关系。我们还建议利用这项新技术来检测损伤部位细胞对不同凝血酶功能的支持作用。通过扩大位点特异性荧光凝血蛋白衍生物的资料库，包括一系列重组小鼠蛋白，我们研究的使能方面将得到加强。认识到在凝血中使用基因操纵的小鼠模型的广泛兴趣，我们认为，我们的策略将使该领域拥有使能技术，将活体成像方法推向新的水平，并允许广泛适用的研究，以破译体内血液凝固途径的调节。印章对当地经济有很大贡献。2008年，CHOP的业务在该地区创造和支持了超过16,882个工作岗位，CHOP的总经济影响超过20.1亿美元。此外，通过私人捐款、NIH资助和医院运营拨款，CHOP获得的研究资助总额超过美国其他任何一家儿童医院--2007-2008财年为1.8亿美元。目前的提案将创造2个就业机会，并为保留2名调查员的工作提供部分支持。在损伤部位形成血栓是一系列凝血反应的结果，这些凝结反应与流动的血液中的血细胞和血管内衬细胞一起发生。我们基于凝血的生物化学知识，寻求通过开发新的荧光方法和试剂来解决在活体中对血液凝结反应进行成像的挑战。这一使能技术将提供必要的资金，以便对正常情况下和威胁生命的人类疾病中血液凝块是如何形成的有新的见解。 与公共卫生相关：在损伤部位形成血块是一系列凝血反应的结果，这些凝血反应与血液中的血细胞和血管内衬细胞同时发生。我们基于凝血的生物化学知识，寻求通过开发新的荧光方法和试剂来解决在活体中对血液凝结反应进行成像的挑战。这一使能技术将提供必要的资金，以便对正常情况下和威胁生命的人类疾病中血液凝块是如何形成的有新的见解。

DESCRIPTION (provided by applicant): We propose to tackle the challenge (06-HL-103) of providing enabling technologies directed towards developing novel quantitative imaging approaches for the measurement of coagulation enzyme function in the intact mouse to improve basic understanding of cellular interactions, the biological pathway for coagulation and its regulation in flowing blood. The intravital fluorescence microscopy approach for examining thrombus formation following laser-induced damage in the cremaster arteriole of the mouse has opened new avenues for quantitative studies of thrombus formation in the blood of a living organism. However, the approach has relied on indirect measures employing fluorescent antibodies to select reactants. Its full promise has been limited by the availability of reagents and approaches for the direct measurement of the individual steps of coagulation. We propose to exploit our established expertise in the biochemistry of the coagulation reactions coupled with an array of well-characterized site-specific fluorescent derivatives of the clotting proteins with defined properties to develop enabling technologies directed towards sensitive and quantitative measurements of coagulation enzyme complex assembly and function at the site of the growing thrombus. We propose approaches designed to examine the spatial relationship between coagulation enzyme localization and the evolving thrombus. We also propose to utilize this new technology to examine the contributions of cells present at the site of injury to differentially support coagulation enzyme function. The enabling aspect of our studies will be enhanced by the expansion of the repertoire of site-specific fluorescent coagulation protein derivatives to include a series of recombinant mouse proteins. Recognizing the widespread interest in the use of genetically manipulated mouse models in coagulation, we submit that our strategies will equip the field with the enabling technologies to move the intravital imaging approach to the next level and permit widely applicable studies to decipher the regulation of the blood coagulation pathways in vivo. CHOP contributes substantially to the local economy. In 2008, CHOP's operations created and supported over 16,882 jobs in the region, and CHOP's total economic impact was over $2.01 billion. Moreover, through a combination of private donations, NIH funding, and allocations from its hospital operations, CHOP receives more total research support than any other children's hospital in the United States -- $180 million in fiscal year 2007-2008. The current proposal will create 2 jobs and provide partial support for the retention of jobs for 2 investigators. The formation of a blood clot at the site of injury results from a series of clotting reactions that occur in concert with blood cells and cells lining the blood vessel in flowing blood. We seek to solve the challenge of imaging the blood clotting reactions in the living organism by the development of novel fluorescent approaches and reagents based on knowledge of the biochemistry of coagulation. This enabling technology will provide the wherewithal for new insights into how blood clots form under normal conditions and in life-threatening human diseases. PUBLIC HEALTH RELEVANCE: The formation of a blood clot at the site of injury results from a series of clotting reactions that occur in concert with blood cells and cells lining the blood vessel in flowing blood. We seek to solve the challenge of imaging the blood clotting reactions in the living organism by the development of novel fluorescent approaches and reagents based on knowledge of the biochemistry of coagulation. This enabling technology will provide the wherewithal for new insights into how blood clots form under normal conditions and in life-threatening human diseases.