Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

Abstract

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Hydrogen‐deuterium exchange mass spectrometry highlights conformational changes induced by factor XI activation and binding of factor IX to factor XIa

“…For Lys 509 ‐Glu 525 , located at the surface of the catalytic domain, this may be caused by a reduced accessibility as a result from movement of the activation loop upon activation. This corresponds to results obtained in a previous study using tandem mass tag mass spectrometry that suggested shielding of the region by the released activation loop after cleavage …”

“…FXI‐WT cDNA with Cys 11 replaced by Ser was introduced in pCDNA3.1 . FXI‐S557A was prepared from FXI wild type (WT) by Quikchange mutagenesis.…”

“…At confluence, serum‐free medium (Optimem/Glutamax 1) was added and collected every 48‐72 hours. FXI was purified from expression medium using a peptide IV column after concentrating the collected medium over an artificial kidney. The peptide IV column was eluted with 100 mmol/L citrate pH 5.0, 1 mol/L NaCl, and 10 mmol/L EDTA.…”

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI

“…A comparison of PKa with the FXI structure revealed a large conformational 180° rotational rearrangement of the intact apple domain disc relative to the protease domain. Low‐resolution structural studies and biochemical data comparing the homologous FXI and active form FXIa have suggested that a large conformational shape change occurs upon activation . Future studies will be required to determine a crystal structure of the PK zymogen to establish whether this mirrors the conformation observed for the FXI zymogen and to determine whether the PKa apple 3 domain is utilized for substrate recruitment as it is in FXIa.…”

Studies into prekallikrein activation pave the way for new avenues of antithrombotic research

“…In addition, multiple groups are studying natural inhibitors of FXIa, FXIIa, and PKa. A phase II clinical trial studying the antisense oligonucleotide demonstrated significantly reduced bleeding in patients undergoing knee arthroplasty, compared with those treated with enoxaparin, supporting the proposed benefits of targeting the contact pathway. These proposed anticontact pathway agents were all designed without the benefit of structural knowledge of FXIa, FXIIa, or PKa.…”