biostudies-literatureUnknown20(11)Bar Barroeta A<h4>Background</h4>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.<h4>Objectives</h4>To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX).<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.Journal of thrombosis and haemostasis : JTH2538-2549https://www.ebi.ac.uk/biostudies/studies/S-EPMC9795894biostudies-literatureNanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.PMC9795894Marquart JAMeijers JCMMeijer ABUrbanus RTBakhtiari KBar Barroeta AfalseNanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.<h4>Background</h4>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.<h4>Objectives</h4>To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX).<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.2022-01-01T00:00:00Z2022 Nov2024-11-19T16:55:23.12Z2024-11-19T16:55:23.12ZS-EPMC97958943581534910.1111/jth.15815