Regulating fibrin polymerization through engineered thermo-responsive knob-pocket

描述（由申请人提供）：拟议的研究旨在开发新技术，使卫生保健专业人员能够更容易地在临床应用中应用纤维蛋白组织密封剂。纤维蛋白是机体在受伤后确保适当体内平衡（防止失血）的天然手段。近一个世纪以来，医生们一直在使用和操纵这种天然止血系统，将其作为一种适用于控制出血和密封组织的聚合物。临床使用的系统由两种天然初级蛋白成分，纤维蛋白原和凝血酶组成，它们是从人类供体中分离纯化的，或者通过重组技术生产的。目前，纤维蛋白是最常用的商业组织密封剂，但尽管其广泛使用，但其显著的局限性继续困扰着该系统的实用性。具体来说，纤维蛋白聚合物在单体活化后形成的速度导致i)在正确应用之前聚合，ii)应用中的可重复性差，以及iii)在许多应用中所需的工作时间不足。在这项研究中，我们建议研究一种新技术，在激活剂（凝血酶）存在的情况下阻断纤维蛋白聚合，直到特定的温度；例如，达到体温。为了实现我们的总体目标，我们提出了两个具体目标。在第一个目标中，我们将创建一个重组蛋白表达系统，使我们能够生产能够显示纤维蛋白阻断元件的蛋白质。这一目标需要当前蛋白质表达系统的基因突变。突变后，我们将把一个模型蛋白克隆到表达系统中，以确定我们是否可以生产出具有关键纤维蛋白阻断元件的蛋白质。然后，我们将测试这种模型蛋白结合非活性纤维蛋白单体（称为纤维蛋白原）的能力，以及它在凝血酶存在下阻断纤维蛋白聚合的能力。在第二个具体目标中，我们将产生热反应蛋白，基于天然蛋白弹性蛋白，包含我们的纤维蛋白阻断元件。这些蛋白质被称为弹性蛋白样肽重复序列或elp，在较低温度下可溶，在较高温度下不溶。我们可以根据肽重复序列和我们在蛋白质中产生的重复次数来定义这些蛋白质从可溶性到不可溶性转变的温度。在这个目标中，我们将生成几个elp，并测试它们结合纤维蛋白原和阻断纤维蛋白聚合的能力，并将测试它们在特定温度下从可溶性到不溶性的转变如何调节它们的结合和阻断活性。设计理念是，当elp变得不溶性时，它们将不再阻碍纤维蛋白聚合，因此在特定温度下，活化的纤维蛋白单体将形成聚合物。拟议的研究将允许开发一种天然存在的基于蛋白质的聚合物系统，该系统可以止血并结合组织，并且在与身体接触时进行独特的聚合。这项新技术不仅可以让医疗保健专业人员更好地控制这个系统，而且还可以开发易于使用的天然液体创可贴产品，广泛应用于公众。

DESCRIPTION (provided by applicant): The proposed study is directed toward the development of novel technology that would enable health care professionals to more readily apply fibrin tissue sealants in clinical applications. Fibrin is the body's natural means for ensuring proper homeostasis (the prevention of blood loss) following injury. For nearly a century, physicians have been using and manipulating this natural hemostatic system as an applicable polymer for controlling bleeding and sealing tissues. The system used clinically consists of the two natural primary protein components, fibrinogen and thrombin, that have been isolated and purified from human donors or produced through recombinant technology. Currently, fibrin is the most used commercially-available tissue sealant but, despite its wide-spread use significant limitations continue to plague the utility of the system. Specifically, the rapidity with which fibrin polymer forms following the activation of the monomer results in i) polymerization prior to proper application, ii) poor reproducibility in application, and iii) insufficient working times required in many applications. In this study we propose researching a novel technology that blocks fibrin polymerization in the presence of the activator (thrombin) until a particular temperature; body temperature for instance, is reached. In order to achieve our overarching goals we have proposed two specific aims. In the first aim, we will create a recombinant protein expression system that allows us to produce proteins that are capable of displaying the fibrin blocking elements. This aim requires the genetic mutation of current protein expression systems. Following mutation we will then clone a model protein into the expression system to determine whether we can produce the protein with the critical fibrin blocking elements. We will then test the capacity of this model protein to bind the inactive fibrin monomer, called fibrinogen, and its capacity to block fibrin polymerization in the presence of thrombin. In the second specific aim we will generate thermo- responsive proteins, based on the natural protein elastin, containing our fibrin blocking elements. These proteins, called elastin-like peptide repeats or ELPs, are soluble at lower temperatures and insoluble at higher temperatures. We can define which temperature these proteins transition from soluble to insoluble based on the sequence of the peptide repeat and the number of repeats we generate in the protein. In this aim we will generate several ELPs and test their capacity to both bind fibrinogen and block fibrin polymerization and will test how their transition from soluble to insoluble at defined temperatures regulates their binding and blocking activities. The design concept is that when the ELPs become insoluble they will no longer block fibrin polymerization, so at specific temperatures activated fibrin monomer will then form a polymer. PUBLIC HEALTH RELEVANCE The proposed study will allow the development of a naturally-occurring protein-based polymer system that stops bleeding and binds tissues and, uniquely, polymerizes in response to contact with the body. This novel technology will not only allow health care professionals better control this system but, it would also allow the development of natural liquid band-aid products that are easy to use with wide-ranging application for the general public.