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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
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Probing activation-driven changes in coagulation factor IX by mass spectrometry.

Nadia Freato1, Floris P J van Alphen1, Mariëtte Boon-Spijker1

  • 1Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands.

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|March 9, 2021
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Summary
This summary is machine-generated.

Activated Factor IX (FIXa) primarily exists in a zymogen-like state. Its enzyme form requires cofactor and substrate interactions, involving the 220-loop and N-terminus, for stabilization.

Keywords:
active sitefactor IXhydrogen-deuterium exchangemass spectrometryserine proteasezymogen

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Area of Science:

  • Biochemistry
  • Proteomics
  • Enzymology

Background:

  • Activated Factor IX (FIXa) is an inefficient enzyme requiring Activated Factor VIII (FVIII) for full activity.
  • Recent studies identified FVIII-driven changes in FIXa using hydrogen-deuterium exchange mass spectrometry (HDX-MS).
  • Some observed changes in FIXa were not cofactor-dependent, particularly within the 220-loop.

Purpose of the Study:

  • To elucidate the zymogen-to-enzyme transition in FIX.
  • To investigate substrate-driven conformational changes at the FIXa catalytic site.

Main Methods:

  • Footprinting mass spectrometry utilizing HDX and Tandem-Mass Tags (TMT) labeling.
  • Site-directed mutagenesis and kinetic analyses to assess the 220-loop's function.

Main Results:

  • HDX-MS revealed minimal differences between FIX and FIXa, with decreased deuterium uptake at the N-terminus in FIXa, especially when inhibited.
  • TMT labeling indicated N-terminus protection, with minor increases upon inhibitor binding.
  • Active site occupation reduced deuterium uptake in the 220-loop backbone.
  • 220-loop mutagenesis identified a hydrogen bond network crucial for FIXa activity, with variants exhibiting more zymogen-like characteristics.

Conclusions:

  • FIXa exists predominantly in a zymogen-like conformation without cofactor and substrate.
  • Stabilization into the enzyme form involves FVIII binding and interactions between the 220-loop, N-terminus, and substrate binding site.