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Published on: May 22, 2020
Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high
Awital Bar Barroeta1, J Arnoud Marquart1, Kamran Bakhtiari1
1Department of Molecular Hematology, Sanquin, Amsterdam, the Netherlands.
Background:
Factor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects.
Objectives:
To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX).
Methods:
Nanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma-based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen-deuterium exchange mass spectrometry.
Results:
We have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI-high molecular weight kininogen (HK) interaction.
Conclusions:
We have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI-FIX interaction.
