VhH anti‐thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow

Abstract

BackgroundThrombomodulin on endothelial cells can form a complex with thrombin. This complex has both anticoagulant properties, by activating protein C, and clot‐protective properties, by activating thrombin‐activatable fibrinolysis inhibitor (TAFI). Activated TAFI (TAFIa) inhibits plasmin‐mediated fibrinolysis. ObjectivesTAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa. In contrast, we here describe variable domain of heavy‐chain‐only antibody (VhH) clone 1 that inhibits TAFI activation by targeting human thrombomodulin. MethodsTwo llamas (Lama Glama) were immunized, and phage display was used to select VhH anti‐thrombomodulin (TM) clone 1. Affinity was determined with surface plasmon resonance and binding to native TM was confirmed with flow cytometry. Clone 1 was functionally assessed by competition, clot lysis, and thrombin generation assays. Last, the effect of clone 1 on tPA‐mediated fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model. ResultsVhH anti‐TM clone 1 bound recombinant TM with a binding affinity of 1.7 ± 0.4 nM and showed binding to native TM. Clone 1 competed with thrombin for binding to TM and attenuated TAFI activation in clot lysis assays and protein C activation in thrombin generation experiments. In a microfluidic fibrinolysis model, inhibition of TM with clone 1 fully prevented TAFI activation. DiscussionWe have developed VhH anti‐TM clone 1, which inhibits TAFI activation and enhances tPA‐mediated fibrinolysis under flow. Different from agents that directly target TAFIa, our strategy should preserve direct TAFI activation via thrombin.