Abstract Number: PB0631
Meeting: ISTH 2022 Congress
Background: Thrombomodulin 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. TAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa, but specific inhibition of TM-mediated TAFI activation has not been attempted.
Aims: To develop variable domain of heavy-chain-only antibody (VhH) clones that inhibit TAFI activation by targeting human thrombomodulin.
Methods: Two Lama Glama were immunized and VhH anti-thrombomodulin clones were selected with phage display. Affinity was determined with Surface Plasmon Resonance and binding to native thrombomodulin was confirmed with flow cytometry. Clone 1 was functionally assessed with competition-, clot lysis- and thrombin generation assays. Lastly, the effect of clone 1 on fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model.
Results: VhH anti-TM clone 1 bound recombinant thrombomodulin with a binding affinity (KD) of 1.7±0.4nM and showed binding to native thrombomodulin. Clone 1 competed with thrombin for binding to thrombomodulin (IC50 148.6nM). Inhibitory effects of clone 1 on thrombomodulin-dependent TAFI activation were confirmed in clot lysis assays: Clone 1 fully attenuated TAFI activation in a dose-dependent manner when thrombomodulin was present, but not in its absence. In addition, clone 1 inhibited protein C activation in thrombin generation experiments in a dose-dependent manner. In a microfluidic fibrinolysis model in which recalcified whole blood was perfused over a collagen, tissue factor and thrombomodulin surface in the presence of tissue plasminogen activator, clone 1 fully prevented TAFI activation.
Conclusion(s): We 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.
To cite this abstract in AMA style:Poolen G, van Moorsel M, Koekman C, Verhoef S, De Maat S, Barendrecht A, van Kleef N, Meijers J, Schiffelers R, Maas C, Urbanus R. VhH anti-thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow [abstract]. https://abstracts.isth.org/abstract/vhh-anti-thrombomodulin-clone-1-inhibits-tafi-activation-and-enhances-fibrinolysis-in-human-whole-blood-under-flow/. Accessed September 22, 2023.
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