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

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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.