Novel Technologies for Imaging Blood Coagulation Reactions

描述（由申请人提供）：我们建议解决提供使能技术的挑战（06-HL-103），该技术旨在开发用于测量完整小鼠中凝血酶功能的新型定量成像方法，以提高对细胞相互作用、凝血生物学途径及其在流动血液中的调节的基本理解。活体荧光显微镜检查血栓形成激光诱导损伤后的小鼠提睾肌小动脉的方法开辟了新的途径，定量研究血栓形成的血液中的一个活的有机体。然而，该方法依赖于采用荧光抗体来选择反应物的间接测量。它的全部承诺受到了试剂和方法的可用性的限制，用于直接测量凝固的各个步骤。我们建议利用我们在凝血反应的生物化学中建立的专业知识，再加上一系列具有明确特性的凝血蛋白的位点特异性荧光衍生物，以开发针对凝血酶复合物组装和功能在生长血栓部位的灵敏和定量测量的技术。我们提出了旨在研究凝固酶定位和血栓形成之间的空间关系的方法。我们还建议利用这种新技术来研究损伤部位细胞对凝血酶功能的差异性支持。我们的研究的使能方面将通过扩展位点特异性荧光凝固蛋白衍生物的库以包括一系列重组小鼠蛋白来增强。认识到在凝血中使用基因操纵的小鼠模型的广泛兴趣，我们提出，我们的策略将配备该领域的使能技术，将活体成像方法提升到一个新的水平，并允许广泛适用的研究，以破译体内凝血途径的调节。CHOP为当地经济做出了巨大贡献。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、国家卫生研究院的资金和医院业务的拨款，CHOP获得的研究资助总额超过美国任何其他儿童医院-2007-2008财政年度为1.8亿美元。目前的提议将创造2个职位，并为保留2名调查员的职位提供部分支助。在受伤部位形成血凝块是由一系列凝血反应引起的，这些凝血反应与流动血液中的血细胞和血管内衬细胞一起发生。我们寻求解决的挑战，成像的血液凝固反应在活的有机体的发展的基础上的知识，凝血的生物化学的新的荧光方法和试剂。这项使能技术将提供必要的手段，以新的见解，血液凝块如何形成在正常条件下，并在危及生命的人类疾病。 公共卫生相关性：在受伤部位形成血凝块是由一系列凝血反应引起的，这些凝血反应与流动血液中的血细胞和血管内衬细胞一起发生。我们寻求解决的挑战，成像的血液凝固反应在活的有机体的发展的基础上的知识，凝血的生物化学的新的荧光方法和试剂。这项使能技术将提供必要的手段，以新的见解，血液凝块如何形成在正常条件下，并在危及生命的人类疾病。

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.