Single-chain factor XII exhibits activity when complexed to polyphosphate

R Engel, C M Brain, Jane Paget, A. S. Lionikiene, N J Mutch

Research output: Contribution to journalArticle

33 Citations (Scopus)
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Abstract

BACKGROUND: The mechanism underpinning factor XII autoactivation was originally characterized with non-physiological surfaces, such as dextran sulfate (DS), ellagic acid, and kaolin. Several 'natural' anionic activating surfaces, such as platelet polyphosphate (polyP), have now been identified.

OBJECTIVE: To analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP70 ).

METHODS AND RESULTS: PolyP70 showed similar efficacy to DS in stimulating autoactivation of FXII, as detected with amidolytic substrate. Western blotting revealed different forms of FXII with the two activating surfaces: two-chain αFXIIa was formed with DS, whereas single-chain FXII (scFXII; 80 kDa) was formed with polyP70 . Dissociation of scFXII from polyP70 abrogated amidolytic activity, suggesting reversible exposure of the active site. Activity of scFXII-polyP70 was enhanced by Zn(2+) and was sensitive to NaCl concentration. A bell-shaped concentration response to polyP70 was evident, as is typical of surface-mediated reactions. Reaction of scFXII-polyP70 with various concentrations of S2302 generated a sigmoidal curve, in contrast to a hyperbolic curve for αFXIIa, from which a Hill coefficient of 3.67 was derived, indicative of positive cooperative binding. scFXII-polyP70 was more sensitive to inhibition by H-d-Pro-Phe-Arg-chloromethylketone and corn trypsin inhibitor than αFXIIa, but inhibition profiles for C1-inhibitor were similar. Active scFXII-polyP70 was also able to cleave its physiological targets FXI and prekallikrein to their active forms.

CONCLUSIONS: Autoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP70 shows activity towards physiological substrates, and may represent the primary event in initiating contact activation in vivo.

Original languageEnglish
Pages (from-to)1513-1522
Number of pages10
JournalJournal of Thrombosis and Haemostasis
Volume12
Issue number9
Early online date13 Aug 2014
DOIs
Publication statusPublished - Sep 2014

Keywords

  • Amino Acid Chloromethyl Ketones
  • Anions
  • Arginine
  • Blood Platelets
  • Catalytic Domain
  • Dextran Sulfate
  • Disulfides
  • Ellagic Acid
  • Enzyme Precursors
  • Factor XII
  • Hemostasis
  • Humans
  • Kaolin
  • Plant Proteins
  • Polyphosphates
  • Prekallikrein
  • Protein Binding
  • RNA
  • Surface Properties

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