Conformational proteins to study molecular mechanisms underpinning GPVI-fibrin(ogen) interaction in blood clot propagation

The formation of a blood clot is a very important normal physiological response to injury. Without adequate clotting, injury could lead to harmful or deadly bleeding. Yet, the flip side of the coin is that too much clotting, or clotting that is too easily triggered, can lead to formation of blood clots that block important blood vessels in the heart (heart attack), brain (stroke), legs (deep vein thrombosis) or lungs (pulmonary embolism). This disease-causing clotting is called thrombosis, which is one of the leading causes of mortality in the developed and developing world. Understandably the process of blood clotting has to be finely regulated to allow for clotting to stem bleeding yet avoid deadly blood clots from developing.

Two key mechanisms are at play during blood clotting. One is the activation of very small blood cells called platelets which aggregate (clump together) and provide the first response during clotting. The second is the conversion of a blood protein called fibrinogen to fibrin by thrombin. Fibrin spontaneously polymerises into a network made of fibres. The fibrin fibre network interacts with the activated platelets to form the blood clot.

It was known that fibrin interacts with platelets through a receptor called GPIIb/IIIa integrin on the platelets. However, new research has shown that there is a second receptor that binds fibrin on the platelet. This receptor is called GPVI and is a member of the immunoglobulin superfamily. The mechanisms by which these two proteins (fibrin and GPVI) interact, and how the interaction contributes to thrombosis, are poorly understood. We have recently found that GPVI binds to fibrin via a particular part of the fibrin molecule, called the alphaC-region. In addition, we have started developing and characterising new conformational proteins called Affimers that are able to bind GPVI and displace fibrin from GPVI and block it from binding.

Our BBSRC research proposal aims to fully characterise the role of GPVI-fibrin interaction in thrombosis using conformational proteins (Affimers) to block this interaction. In the first part of our research project, we will screen a very large number of Affimers for binding to GPVI. Next, we will screen whether the positive binding Affimers block the interaction between GPVI and fibrin. In the second part, we will test blocking Affimers using in-vitro models of platelet aggregation (clumping), thrombus formation and thrombus growth. In the third part, we will characterise the interaction of the Affimers and GPVI at a very detailed molecular level using mass spectrometry, site-directed mutagenesis and X-ray crystallography methods. This will help us to understand how the Affimer interacts with GPVI, and indirectly how fibrin interacts with GPVI. Together, these studies will enhance our understanding of this important novel pathway of thrombus formation and may help to find new ways of preventing deadly blood clots in patients at risk of a heart attack, stroke, deep vein thrombosis or pulmonary emblism.

Thrombosis is a leading cause of morbidity and mortality worldwide. Key contributors to thrombosis are platelets that activate and aggregate, in combination with fibrin which is produced by thrombin-mediated cleavage of fibrinogen. Fibrin and platelets were known to interact via integrin receptor GPIIb/IIIa. Recent data have shown a novel secondary pathway via which these two key players interact and contribute to thrombosis, involving the binding of fibrin to platelet receptor GPVI. The molecular mechanisms via which these two proteins interact and contribute to thrombosis are however poorly understood. We recently identified the fibrin alphaC-region as a key site for GPVI interaction. Furthermore, we have obtained key pilot data for the generation of conformational proteins (Affimers) that bind GPVI and will test whether these inhibit its interaction with fibrin. The aim of this proposal is to screen and characterise Affimers that inhibit GPVI-fibrin interaction in order to elucidate new molecular mechanisms that drive thrombosis. Specifically, we will: 1) Identify Affimers that bind GPVI and inhibit GPVI from interacting with fibrin using plate-based binding assays, surface plasmon resonance, microscale thermophoresis and competition assays, 2) Characterise the effect of GPVI-fibrin interaction blocking Affimers on platelet aggregation, platelet spreading and thrombus formation and propagation under flow, and 3) Elucidate molecular details of GPVI-fibrinogen/Affimer interaction sites using mass spectrometry, site-directed mutagenesis and X-ray crystallography. These studies will help to elucidate novel molecular mechanisms that contribute to thrombosis, which may underpin the future development of new anti-thrombotic treatments with minimal bleeding risk.