Use of Affimer Technology for Inhibition of α2-antiplasmin and Enhancement of Fibrinolysis

• Function of antifibrinolytic protein α2AP can be modulated using Affimers, facilitating lysis of clots from plasma and whole blood samples

• α2AP-specific Affimer enhanced clot lysis through a dual mode of action by increasing both plasmin activity and generation

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP) and we hypothesise that the Affimer technology, comprising small conformational proteins with two nine amino acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis. Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated of which one, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n=15; from 150.8±100.9 to 109.8±104.8 mins) and those with cardiovascular disease (n=15; 117.6±40.6 to 79.7±33.3 mins); p<0.01 for both groups. The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples. Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), while molecular modelling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15-0.60 mg/ml, had the ability to bind 70-90% of plasma α2AP. In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.