Dataset Information

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

ABSTRACT:

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.

SUBMITTER: Bar Barroeta A

PROVIDER: S-EPMC9795894 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

Bar Barroeta Awital A Marquart J Arnoud JA Bakhtiari Kamran K Meijer Alexander B AB Urbanus Rolf T RT Meijers Joost C M JCM

Journal of thrombosis and haemostasis : JTH 20220720 11

<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 ...[more]

PMID: 35815349

Similar Datasets

Project description:Essentials Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N-terminal apple domains to recruit substrates. A high-resolution 1.3 Å structure of full-length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four-apple domain disc organization is 180° rotated compared to FXI. SUMMARY: Background Plasma prekallikrein (PK) and factor XI (FXI) are apple domain-containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. Objective To investigate the three-dimensional structure of full-length PKa and perform a comparison with FXI. Methods A series of recombinant full-length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusions A 1.3 Å structure of full-length PKa reveals the protease domain positioned above a disc-shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen-deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.

Dataset Information

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

Background

Objectives

Methods

Results

Conclusions

Publications

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

Similar Datasets

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