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

纤维蛋白原靶向构象蛋白用于鉴定控制纤维蛋白网络稳定性的机制途径

• 批准号: BB/T013583/1
• 负责人: Ramzi Ajjan
• 金额: $ 68.58万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT013583%2F1
• 关键词: Fibrinogen; targeted; conformational; proteins; identification

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.

这项工作的目的是什么？血管受伤后，会形成血栓，这对于限制失血和挽救生命至关重要。严重的出血事件，威胁生命，可能发生在事故，外科手术后，在一些人中，他们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，以确保不必要的血栓不会形成，但这些仍然可能发生阻塞血管，导致心脏病发作，中风和深静脉血栓形成。了解凝块形成和分解的细节将有助于充分了解这一过程，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。使用一种新技术（由我们的团队开发），目前的项目我们将使用小蛋白质，称为Affimers，来阻止血液凝块形成和分解的过程。作为在这一领域进行的研究的结果，我们已经知道了什么？血凝块由纤维蛋白纤维网组成，血细胞被困在这个网络中。这种网状物形成了血凝块的骨架，它对分解的敏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一种丰富的血浆蛋白（称为纤维蛋白原）形成的，并由许多其他蛋白质稳定下来。我们的初步数据，发表在著名的杂志“血液”，表明我们已经开发的Affimer蛋白质，并且是特定的纤维蛋白原，能够改变血液凝块分解的容易程度，使它们成为可以帮助研究血液凝块抵抗分解的精细细节的试剂。我们希望通过三个明确的步骤将工作推向下一个层次：i）分离大量结合纤维蛋白原并增加抵抗力或促进血凝块分解的Affimers，ii）广泛测试Affimers稳定纤维蛋白网或增加其对分解的敏感性的（机制），这将使我们能够确定适合开发新药的新靶点，和iii）在合适的小鼠动物模型中分析感兴趣的亲和聚体的作用。这将为我们提供一个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的指示。我们将如何开展这项工作？拟议的工作涉及我们实验室已经优化的最先进的研究技术。我们有大约30亿种不同的Affimers，将对其与纤维蛋白原的结合和干扰血栓分解进行分析。新分离的Affimers和我们已经确定的其他Affimers将使用来自健康人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确定功能至关重要，从而导致凝块更稳定或更容易分解。在这项工作的最后一部分，我们将在小鼠中进行动物研究，以确认在试管（体外）中发现的Affimers的效果在体内进行实验时不会丢失。这项研究如何有益？这项工作产生的数据将确定Affimers在蛋白质功能研究中的作用，有助于理解正常生理学，并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，以减少高危人群的出血或不必要的血管闭塞（血栓形成）。

项目成果

Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges.

• DOI: 10.3390/ijms22136916
• 发表时间: 2021-06-28
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Gaule TG;Ajjan RA
• 通讯作者: Ajjan RA

Fibrinogen and Antifibrinolytic Proteins: Interactions and Future Therapeutics.

• DOI: 10.3390/ijms222212537
• 发表时间: 2021-11-21
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Pechlivani N;Kearney KJ;Ajjan RA
• 通讯作者: Ajjan RA

Current and novel biomarkers of thrombotic risk in COVID-19: a Consensus Statement from the International COVID-19 Thrombosis Biomarkers Colloquium.

• DOI: 10.1038/s41569-021-00665-7
• 发表时间: 2022-07
• 期刊: Nature reviews. Cardiology
• 作者: Gorog DA;Storey RF;Gurbel PA;Tantry US;Berger JS;Chan MY;Duerschmied D;Smyth SS;Parker WAE;Ajjan RA;Vilahur G;Badimon L;Berg JMT;Cate HT;Peyvandi F;Wang TT;Becker RC
• 通讯作者: Becker RC

Affinity purification of fibrinogen using an Affimer column.

使用 Affimer 柱对纤维蛋白原进行亲和纯化。

• DOI: 10.1016/j.bbagen.2022.130115
• 发表时间: 2022
• 期刊: Biochimica et biophysica acta. General subjects
• 作者: Pechlivani N