Engineering fibrin polymers for enhanced angiogenesis

描述(申请人提供)：止血，或防止失血，和组织封闭对于应对血管损伤至关重要，无论这些损伤是由于创伤事故还是手术中的计划损伤造成的。虽然目前市场上有几种生物材料可以满足这一需求，但基于纤维蛋白原聚合的人体自身凝血系统仍然是最好的方法。自20世纪初以来，外科医生一直使用血浆纤维蛋白原配方来预防出血；然而，直到蛋白质纯化方面取得进展，基于纤维蛋白原的聚合物系统才在20世纪90年代初开始商业化。今天，基于纤维蛋白原的产品是外科医生止血和组织封闭的压倒性选择，但这些系统并不是没有缺陷。特别是，商业上可获得的纤维蛋白原配方形成的聚合物在性质上没有指导性，也不允许快速伤口修复。这些糟糕的行为主要归因于聚合物缺乏整合素特定的配体和适当的孔隙率。纤维蛋白聚合物能够结合许多整合素，主要是炎症细胞；然而，缺乏细胞与纤维蛋白聚合物结合的特异性会导致过度增殖，而不是有组织的组织重建。此外，尽管聚合物支持早期组织封闭所需的机械载荷，但它缺乏有效血管形成所需的孔洞。在这项申请中，我们建议开发一种新技术，该技术可能使医生能够针对特定的临床结果(例如血管生成)设计他们的纤维蛋白聚合物。我们的中心假设是，通过分别与整合素特异性配体和聚乙二醇相关联的纤维蛋白结合肽，对纤维蛋白聚合物进行生化和物理修饰，将产生增强的血管生成反应。我们将通过四个具体目标来解决这一假设。目标1需要开发与纤维蛋白原和纤维蛋白特异结合的多肽基序。在特定的目标2中，我们将开发一种“即插即用”的表达系统，允许快速生产显示纤维蛋白结合肽的蛋白质。我们将通过体外血管生成试验来探索这些蛋白质对纤维蛋白聚合、生化特性和血管生成潜力的影响。然后，在目标3中，我们将修饰各种形式的聚乙二醇，以展示纤维蛋白结合肽，以改变聚合物结构，并确定这些结合物对纤维蛋白聚合物结构和血管生成潜力的影响。最后，在目标4中，我们将在体内测试我们修改后的纤维蛋白系统。我们的研究结果将对基于纤维蛋白的生物材料的设计产生重大影响，并加强纤维蛋白聚合物在伤口愈合、组织修复和再生医学中的临床应用。 公共卫生相关性：这项拟议的项目解决了以纤维蛋白为基础的生物材料的基本控制，以增强血管生成或新血管形成。该项目将通过建立新的方法来处理特定临床结果的纤维蛋白，从而与伤口愈合和再生医学直接相关。

DESCRIPTION (provided by applicant): Hemostasis, or the prevention of blood loss, and tissue sealing are of critical importance in response to vascular injury, whether those injuries are a result of traumatic accidents or planned injuries as in the case of surgery. While several biomaterials are currently on the market to address this need, the body's own coagulation system based on the polymerization of fibrinogen, still represents the best approach. Since the early 20th century surgeon's have used formulations of plasma fibrinogen to prevent hemorrhage; however, it was not until advances in protein purification were made that fibrinogen-based polymer systems became commercially available, in the early 1990s. Today, fibrinogen-based products are the overwhelming choice of surgeons for hemostasis and tissue sealing, yet these systems are not without their flaws. In particular, commercially available fibrinogen formulations form polymers that are not instructive in nature, nor are they permissive to rapid wound repair. These poor behaviors are primarily attributed to the polymer's lack of integrin-specific ligands and proper porosity. A fibrin polymer is capable of engaging many integrins on mostly inflammatory cells; however the lack of specificity with which cells engage fibrin-based polymers leads to hyperproliferation, rather than organized tissue remodeling. Furthermore, while the polymer supports the mechanical loads necessary for early tissue sealing, it lacks the porosity necessary for efficient vascularization. In this application we propose the development of a novel technology that could potentially enable physicians to design their fibrin polymers for specific clinical outcomes (e.g. angiogenesis). Our central hypothesis is that enhanced angiogenic responses will be elicited from fibrin polymers modified both biochemically and physically with fibrin binding peptides associated with integrin-specific ligands and polyethylene glycol (PEG), respectively. We will address this hypothesis through four specific aims. Aim 1 entails developing peptide motifs that specifically bind fibrinogen and fibrin. In specific aim 2 we will develop a 'plug-and-play' expression system that allows for the rapid production of proteins displaying the fibrin-binding peptides. We will explore the effect of these proteins on fibrin polymerization, biochemical characteristics and angiogenic potential using an in vitro angiogenesis assay. We will then, in Aim 3, modify various forms of PEG to display fibrin binding peptides in order to alter polymer structure and determine the effects of these conjugates on fibrin polymer structure and angiogenic potential. Finally, in Aim 4 we will test our modified fibrin system in vivo. The results of our study will have a significant impact on fibrin-based biomaterials design and enhance the clinical uses of fibrin polymers for wound healing, tissue repair, and regenerative medicine. PUBLIC HEALTH RELEVANCE: This proposed project addresses the fundamental control of fibrin-based biomaterials for enhance angiogenesis, or new blood vessel formation. This project will have direct relevance to wound healing and regenerative medicine by establishing new methods to manipulate fibrin for specific clinical outcomes.