OC 70.4 - Thrombin activation of the factor XI dimer is a multi-staged process for each subunit

Background: 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.

Aims: To identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation.

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).

Results: We identified a binding interface of thrombin located on the light chain of FXI. After this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to the apple 1 domain in a secondary step to allow for migration towards the FXI cleavage site. The 1C10 binding site to the apple 1 domain supports this proposed trajectory of thrombin.

Conclusion(s): Our investigations show that the activation of FXI is a multi-staged procedure. Thrombin first binds to Pro520 in FXI, after which it migrates towards the activation site by first engaging the apple 1 domain and, finally, Arg378. We further 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. This detailed analysis of the interaction between thrombin and FXI points a way for future interventions for bleeding or thrombosis.