Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor

Background and objective: As activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid-derived single-domain antibodies towards TAFI. Methods and results: This study reports the generation and characterization of a panel of 22 inhibitory nanobodies. This panel represents a wide diversity in mechanisms for interference with the functional properties of TAFI as the nanobodies interfere with various modes of TAFI activation, TAFIa activity and/or TAFI zymogen activity. Nanobodies inhibiting TAFIa activity and thrombin/thrombomodulin-mediated TAFI activation revealed pro. brinolytic properties in a clot lysis experiment with exogenously added thrombomodulin (TM), whereas nanobodies inhibiting plasmin-mediated TAFI activation only revealed pro. brinolytic properties in a clot lysis experiment without TM. The results of in vitro clot lysis experiments provided evidence that inhibitory nanobodies penetrate the clot better compared with inhibitory monoclonal antibodies. Conclusions: These data suggest that the generated nanobodies are potent TAFI inhibitors and are a step forward in the development of a pro. brinolytic drug. They might also be an excellent tool to unravel the role of the physiological activators of TAFI in various pathophysiological processes.status: publishe