PB0631 - VhH anti-thrombomodulin clone 1 inhibits TAFI activation and enhances fibrinolysis in human whole blood under flow

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Geke C. Poolen, MSc

PhD student
Center for Benign Haematology, Thrombosis and Haemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, The Netherlands
Utrecht, Utrecht, Netherlands

Co-Author(s)

Mv

Marc van Moorsel, MSc

PhD Candidate
UMC Utrecht
Utrecht, Utrecht, Netherlands
CK

Cornelis Koekman

Head Research Technician
University Medical Center Utrecht
Utrecht, Utrecht, Netherlands
SV

Sandra Verhoef

Research technician
University Medical Center Utrecht
Utrecht, Utrecht, Netherlands
Steven de Maat, PhD

Assistant Professor
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Utrecht, Utrecht, Netherlands
AB

Arjan D. Barendrecht

Lab technician
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
Nv

Nadine D. van Kleef

Research technician
University Medical Center Utrecht
Utrecht, Utrecht, Netherlands
JM

Joost CM Meijers

Biochemist
Department of Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
RS

Raymond Schiffelers

Professor of Nanomedicine
CDL Research, University Medical Center Utrecht
Utrecht, Utrecht, Netherlands
Coen Maas, PhD

Associate Professor
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
RU

Rolf T. Urbanus

Associate professor
Center for Benign Haematology, Thrombosis and Haemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
Utrecht, Utrecht, Netherlands

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.