Probing activation-driven changes in coagulation factor IX by mass spectrometry

Nadia Freato, Floris P J van Alphen, Mariëtte Boon-Spijker, Maartje van den Biggelaar, Alexander B Meijer, Koen Mertens, Eduard H T M Ebberink

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

BACKGROUND: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering).

OBJECTIVE: The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site.

METHODS: Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop.

RESULTS: HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa.

CONCLUSION: In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site.

Original languageEnglish
Pages (from-to)1447-1459
Number of pages13
JournalJournal of Thrombosis and Haemostasis
Volume19
Issue number6
DOIs
Publication statusPublished - Jun 2021

Keywords

  • zymogen
  • serine protease
  • mass spectrometry
  • hydrogen-deuterium exchange
  • factor IX
  • active site

Fingerprint

Dive into the research topics of 'Probing activation-driven changes in coagulation factor IX by mass spectrometry'. Together they form a unique fingerprint.

Cite this