Fibrinogen-targeted conformational proteins for identification of the mechanistic pathways controlling fibrin network stability

这项工作的目的是什么？血管受伤后，会形成血凝块，这对限制失血和维持生命至关重要。严重出血事件，威胁生命，可能发生在事故和外科手术之后，对一些人来说，这可能是由于罕见的遗传疾病，使他们在轻微创伤后出血。虽然有一个很好的平衡，以确保不需要的血凝块不会形成，但这些仍然会阻塞血管，导致心脏病发作、中风和深静脉血栓形成。了解血栓形成和破裂的细节将有助于充分描述这一过程，这反过来将有助于发现新的靶点，用于开发减少受伤后出血的药物，或者相反，限制不必要的血管阻塞，以防止心脏病发作、中风和深静脉血栓形成。使用一项新技术（由我们的团队开发），我们目前的项目将使用一种叫做Affimers的小蛋白质来描述血凝块形成和分解的过程。通过这一领域的研究，我们已经知道了什么？血凝块是由纤维蛋白纤维网组成的，血细胞被困在这个网络中。这种网状结构构成了血凝块的骨架，它对破裂的易感性可以决定一个人出血或血管阻塞的风险。这种纤维蛋白网是由丰富的血浆蛋白（称为纤维蛋白原）形成的，并由许多其他蛋白质结合到这种网中来稳定。我们发表在著名期刊《血液》上的初步数据显示，我们开发的纤维蛋白原特异性的Affimer蛋白能够改变血凝块分解的容易程度，使它们成为有助于研究血凝块抵抗分解的细节的试剂。我们希望把定义的工作到下一个水平三个步骤:1)分离大量Affimers结合纤维蛋白原和增加阻力或促进血凝块的分解,ii)广泛测试的方法(机制)Affimers稳定纤维蛋白网或增加其容易崩溃,这将使我们能够确定新的目标适合开发新药物,和3)分析的影响Affimers兴趣的合适的小鼠动物模型。这将给我们指明新发现的靶点是否适合未来使用新一代药物进行治疗操作。我们将如何开展这项工作？提议的工作涉及已经在我们的实验室优化的最先进的研究技术。我们有大约30亿个不同的确定子，它们将被分析与纤维蛋白原的结合和对凝块分解的干扰。新分离的Affimers和我们已经确定的其他Affimers将使用健康人群以及具有过度出血或形成血栓倾向的临床症状的个体的血液样本进行测试。随后将进行实验，试图了解纤维蛋白原蛋白上的哪些区域对决定功能至关重要，从而导致更稳定或更容易分解的凝块。在工作的最后一部分，我们将在小鼠身上进行动物研究，以确认在试管（体外）中发现的Affimers的作用在体内进行实验时不会消失。这项研究有什么好处？从这项工作中产生的数据将确定确认子在蛋白质功能研究中的作用，有助于理解正常生理学和揭示一些病理条件背后的机制。从长远来看，这将有助于确定纤维蛋白原上改变蛋白质功能的新区域，并可用于开发新一代治疗药物，以减少高风险人群的出血或不必要的血管闭塞（血栓形成）。

What is the aim of this work?After injury to a blood vessel, a blood clot is formed which is critical to limit blood loss and preserve life. Severe bleeding events, threatening life, may occur following accidents, surgical operations and in some people they may be due to rare genetic diseases that make them bleed after minor trauma. While there is a fine balance to make sure unwanted blood clots do not form, these can still occur obstructing a blood vessel and causing heart attacks, strokes and deep venous thrombosis. Understanding the details of clot formation and breakdown will help to fully characterise the process, which in turn will enable the discovery of new targets for the development of drugs that reduce bleeding after injury or, conversely, limit unwanted blood vessel obstruction in order to protect from heart attacks, strokes and deep venous thrombosis. Using a new technology (developed by our team), the current project we will use small proteins, called Affimers, to characterise the process of blood clot formation and breakdown.What do we already know as a result of research carried out in this area?The blood clots is composed of a mesh of fibrin fibres with blood cells trapped in this network. This mesh forms the skeleton of the blood clot and its susceptibility to breakdown can determine someone's risk of bleeding or blood vessel obstruction. This fibrin mesh forms from an abundant plasma protein, called fibrinogen, and is stabilised by a number of other proteins that get incorporated into this mesh. Our preliminary data, published in the prestigious journal "Blood", show that Affimer proteins that we have developed, and that are specific to fibrinogen, are able to modify how easily the blood clot is broken down, making them agents that can help to study the fine details of blood clot resistance to breakdown. We wish to take the work to the next level in three defined steps: i) isolate a large number of Affimers that bind fibrinogen and either increase resistance or facilitate breakdown of the blood clot , ii) extensively test the ways (mechanisms) by which Affimers stabilise the fibrin mesh or increase its susceptibility to breakdown, which will allow us to identify new targets suitable for developing new drugs, and iii) analyse the effects of Affimers of interest in suitable mouse animal models. This will give us an indication of the suitability of the newly discovered targets for future therapeutic manipulation using a new generation of drugs. How will we carry out the work?The proposed work involves state-of-the-art research techniques that have already been optimised in our laboratories. We have around 3 billion different Affimers that will be analysed for binding to fibrinogen and for interfering with clot breakdown. Newly isolated Affimers, and others which we have already identified, will be tested each using blood samples from healthy people as well as individuals with clinical conditions characterised by excessive bleeding or a tendency to form blood clots. This will be followed by experiments trying to understand which areas on the fibrinogen protein are critical to determine function, thereby leading to clots that are more stable or easier to breakdown. In the final strand of the work, we will conduct animal studies in mice to confirm that the effects of Affimers found in the test tube (in vitro) are not lost when experiments are conducted in vivo. How is this research beneficial?Data generated from this work will establish the role of Affimers for the study of protein function, helping to understand normal physiology and shedding light on the mechanisms behind some pathological conditions. In the long-run, this will help to identify novel areas on fibrinogen that alter protein function and can be used to develop a new generation of therapeutic agents for the reduction of bleeding or unwanted blood vessel occlusion (thrombosis) in high risk people.