The use of plasminogen activator inhibitor-1 specific Affimers as a tool to modulate fibrin clot properties and thrombosis risk

Abstract

The ability to form a blood clot is critical for preventing blood loss following vessel injury. After restoring vascular integrity, the haemostatic system allows for the closure of impaired blood vessels, the preservation of blood in a fluid state, and the removal of clots. Blood clots are made up of a network of fibrin fibres that include platelets, red blood cells, and white blood cells. Imbalances and changes in coagulation factors, as well as the several components of the coagulation cascade, may have an effect on fibrinolysis. This turn may lead to increased risk of unwanted vascular occlusion through the failure to remove intravascular clots and maintain blood flow, consequently resulting in reduced blood supply to an organ. Hypofibrinolysis is a key abnormality in conditions associated with high vascular risk such as diabetes, contributing to the adverse vascular outcome in this population. Plasminogen activator inhibitor (PAI)-1 is an important regulator of the fibrinolytic process and levels of this antifibrinolytic protein are elevated in diabetes and insulin resistant states. An increase in PAI-1 levels results in impaired fibrinolysis and increases the risk of thrombus formation. Therefore, modulating PAI-1 activity has the potential to improve the fibrinolytic process and decrease intravascular clot formation. One difficulty with modulating PAI-1 function is the reliance of this protein on vitronectin that stabilises PAI-1 and enhances its function. I hypothesised that plasminogen activator inhibitor-1 specific Affimers, which are small synthetic proteins, may be used to modulate fibrinolysis, consequently reducing the risk of thrombosis. The main aims of my work were to i) isolate PAI-1- specific Affimers, ii) investigate the effects of PAI-1-specific Affimers on fibrinolysis, and iii) characterise the mechanistic pathways for Affimer-mediated modulation of clot lysis. My work shows that PAI-1 binding Affimers do not adequately modulate protein function and only Affimers that bind both PAI-1 and vitronectin demonstrate an ability to significantly alter both protein function and consequently the fibrinolytic process. I also explore the mechanistic pathways that are responsible for Affimer-mediated PAI- 1 inhibition, in the hope that this may help to develop effective therapies to reverse the hypofibrinolytic environment in high vascular risk conditions.