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

Factor XI (FXI) is a protein in the intrinsic coagulation pathway that 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 poorly understood due to their transient nature which is difficult to study. Here, we applied crosslinking mass spectrometry (XLMS) and molecular dynamics simulations to investigate the binding interface between thrombin and FXI as well as the dynamics underlying the activation of FXI. From the experimental data we were able to construct the FXI homodimer as well as locate the binding interface of thrombin on the light chain of FXI. As this placed thrombin distal from both the activation site as well as the apple 1 domain that thrombin is known to interact with, we applied molecular dynamics simulations to investigate the chain of events after binding. From the resulting model, we hypothesize that the activation of FXI is a multi-staged procedure where thrombin first binds to Pro520 on FXI, after which it migrates towards the activation 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 and enables binding of thrombin to this enzyme even though it cannot be activated by it. This interaction points a way for future interventions for bleeding and thrombosis.