Awital Bar Barroeta

Thrombin activation of the factor XI dimer is a multi-staged process for each subunit

Awital Bar Barroeta1, Pascal Albanese2,3, J. Arnoud Marquart1, Joost C.M. Meijers1,4, Richard A. Scheltema2,3

1 Department of Molecular Hematology, Sanquin, Amsterdam, the Netherlands
2 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
3 Netherlands Proteomics Centre, Utrecht, The Netherlands
4 Department of Experimental Vascular Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

Introduction: Factor XI (FXI), a protein in the intrinsic coagulation pathway, can be activated by two enzymes. In hemostasis, FXI is activated by thrombin, while FXIIa-mediated activation of FXI is thought to be prothrombotic. The interactions of these enzymes with FXI are transient in nature and therefore difficult to study in a structural context.

Methods: Crosslinking mass spectrometry (XLMS) was used to construct the FXI homodimer and to localize the binding interface of thrombin on FXI. Molecular dynamics simulations were then applied to investigate conformational changes after binding. Additionally, the binding site of nanobody 1C10 – previously shown to inhibit thrombin-mediated activation of FXI – was investigated with hydrogen-deuterium exchange mass spectrometry (HDX MS).

Preliminary data: Our investigations show that the activation of FXI is a multi-staged procedure. We identified that thrombin initially interacts with the light chain of FXI by binding Pro520. Following this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to allow migration towards the FXI cleavage site by first engaging the apple 1 domain and, finally, Arg378. We validated the results with known mutation sites on FXI and additionally found that Pro520 is conserved in PK. Through this site, thrombin can bind PK even though it cannot activate PK. Moreover, the proposed trajectory of thrombin-mediated FXI activation was supported by elucidation of the 1C10 binding site on the apple 1 domain. This detailed analysis of the interaction between thrombin and FXI points a way for future interventions for bleeding or thrombosis.

Novel aspect: Elucidation of the transient enzymatic thrombin-FXI interaction and the corresponding activation mechanism.

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