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

30 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

Fingerprint

Factor XII
Polyphosphates
Dextran Sulfate
Blood Platelets
prolyl-phenylalanyl-arginine-4-nitroanilide
Prekallikrein
Ellagic Acid
Kaolin
Catalytic Domain
Western Blotting

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

Cite this

Single-chain factor XII exhibits activity when complexed to polyphosphate. / Engel, R; Brain, C M; Paget, Jane; Lionikiene, A. S.; Mutch, N J.

In: Journal of Thrombosis and Haemostasis, Vol. 12, No. 9, 09.2014, p. 1513-1522.

Research output: Contribution to journalArticle

Engel, R ; Brain, C M ; Paget, Jane ; Lionikiene, A. S. ; Mutch, N J. / Single-chain factor XII exhibits activity when complexed to polyphosphate. In: Journal of Thrombosis and Haemostasis. 2014 ; Vol. 12, No. 9. pp. 1513-1522.
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T1 - Single-chain factor XII exhibits activity when complexed to polyphosphate

AU - Engel, R

AU - Brain, C M

AU - Paget, Jane

AU - Lionikiene, A. S.

AU - Mutch, N J

N1 - © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

PY - 2014/9

Y1 - 2014/9

N2 - 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.

AB - 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.

KW - Amino Acid Chloromethyl Ketones

KW - Anions

KW - Arginine

KW - Blood Platelets

KW - Catalytic Domain

KW - Dextran Sulfate

KW - Disulfides

KW - Ellagic Acid

KW - Enzyme Precursors

KW - Factor XII

KW - Hemostasis

KW - Humans

KW - Kaolin

KW - Plant Proteins

KW - Polyphosphates

KW - Prekallikrein

KW - Protein Binding

KW - RNA

KW - Surface Properties

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DO - 10.1111/jth.12663

M3 - Article

VL - 12

SP - 1513

EP - 1522

JO - Journal of Thrombosis and Haemostasis

JF - Journal of Thrombosis and Haemostasis

SN - 1538-7933

IS - 9

ER -