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

Reference	BB/T013583/1
Principal Investigator / Supervisor	Professor Ramzi Ajjan
Co-Investigators / Co-Supervisors	Professor Robert Ariens, Dr Thomas Edwards, Professor Khalid Naseem, Dr V Ridger, Professor Robert Storey, Dr Christian Tiede, Dr Darren Tomlinson
Institution	University of Leeds
Department	School of Medicine
Funding type	Research
Value (£)	538,220
Status	Current
Type	Research Grant
Start date	01/03/2021
End date	29/02/2024
Duration	36 months

Abstract

The fibrin network represents the backbone of the blood clot and its susceptibility to breakdown determines predisposition to bleeding and thrombosis. Fibrin clots are made from the abundant plasma protein fibrinogen and stabilised by factor (F)XIII-mediated incorporation of anti-fibrinolytic proteins into the fibrin networks, including plasmin inhibitor (PI), one of the most potent proteins protecting clot breakdown. Affimers are small conformational proteins composed of a scaffold protein and two variable regions. These proteins are characterised by great diversity and ease of production in large quantities. We hypothesise that Affimers represent a tool to understand the functional properties of fibrinogen and uncover novel therapeutic targets to modulate clot susceptibility to lysis. Our preliminary data have already shown feasibility of such an approach and the current proposal has three objectives: i) isolate additional fibrinogen-specific Affimers that modulate fibrinolysis, ii) fully characterise existing and newly isolated Affimers, and iii) test Affimers of interest using in vivo models of bleeding and thrombosis. Affimers will be isolated using phage display techniques and characterised using functional assays, including turbidimetric analysis, FXIII activity and PI incorporation assays, thrombin generation and fibrinopeptide release analyses. Fibrinogen-Affimer interaction sites will be determined using crystallography, microarray analysis, pull down assays and molecular modelling. Animal ex vivo and in vivo studies will ensure that Affimers stabilising the fibrin network are able to control bleeding while those that enhance clot breakdown limit vessel occlusion. Data generated from this work will establish the role of Affimers as a convenient tool to study protein function and protein-protein interactions. In the long-run, this will help to identify novel therapeutic targets for the reduction of bleeding or thrombosis in high risk individuals.

Summary

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.

Impact Summary

Formation of a stable blood clot is essential to prevent life-threatening bleeding after trauma. On the other hand, unwanted blood clots can form in diseased blood vessels that can result in organ damage (heart attack or stroke) and can even lead to death. The blood clot is composed of a skeleton of fibrin fibres with blood cells trapped in this network. This mesh of fibrin fibres forms from a plasma protein called fibrinogen and is further stabilised by incorporating other plasma proteins into the network. Understanding the mechanisms that govern fibrin network formation and its resistance to breakdown will have important scientific implications, including understanding the mechanisms for increased bleeding or thrombosis risk. The current proposal explores a new methodology to modulate fibrin network formation and breakdown, using small proteins called Affimers. This project has the potential to impact academia, economy, industry, public engagement and provision of highly skilled people. Scientific impact The proposed work will employ Affimers to understand the functional areas on fibrinogen that control fibrin clot stability. This will provide the academic coagulation community with unprecedented data on the contribution of different areas of the fibrinogen molecule to fibrin clot stability and resistance to breakdown. It will also provide key information on the interaction between fibrinogen and one of the most potent anti-fibrinolytic factors, plasmin inhibitor. Data generated may uncover novel therapeutic targets that can be developed to aid in the management of bleeding or thrombotic conditions. Moreover, the academic benefits may extend beyond coagulation proteins, as Affimers may become a useful tool to study protein function in other areas of research. Economic and social impact Both bleeding and thrombotic disorders are important causes of mortality and ill health, consequently affecting quality of life (QoL) and the productivity of an individual. In depth understanding of the mechanistic pathways operating in fibrin clot formation and stability will help to probe into the factors determining excessive bleeding or thrombosis. This in turn will help in the future to better identify individuals at risk who can be offered alternative management strategies, thus improving health, QoL and productivity. Development of academia-industry partnership The work has the potential to introduce new understanding of the mechanisms involved in fibrin clot formation/breakdown and may uncover new therapeutic targets that alter stability of the fibrin network. This will be of interest to industry, particularly if the introduced changes in clot stability can be optimised to reduce bleeding without increasing thrombosis risk (and vice versa). The University of Leeds has mechanisms in place to support the exploitation of intellectual property arising from this project and can engage relevant commercial partners for future development of therapeutics. Public engagement We have a strong track record of active participation in public understanding of science activities, which includes exploring research concerns of the public. The Principal Investigator has direct links with patient and public involvement groups and he has constant input from the public into his research. Development of highly skilled people We will train the Postdoctoral Fellow and the Research Technician on the grant by providing a multidisciplinary research environment and improving on a range of molecular biology, biophysical, biochemical and essential animal work within the context of understanding the functional properties of the fibrinogen molecule. We will explore opportunities for gaining PhD studentship in 2020 to be associated with the project, which will strengthen the team and provide opportunities for further research training.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology, Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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