Polyphosphate binds with high affinity to exosite II of thrombin

N J Mutch, T Myles, L L K Leung, J H Morrissey

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C-terminal dodecapeptide and γ-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na+-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (Kd approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nmKd for the polyphosphate–thrombin interaction.
Original languageEnglish
Pages (from-to)548-555
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume8
Issue number3
Early online date11 Dec 2009
DOIs
Publication statusPublished - Mar 2010

Fingerprint

Polyphosphates
Thrombin
Heparin
Polymers
Binding Sites
Hirudins
Factor V
Antithrombins
Surface Plasmon Resonance
Platelet Activation
Prothrombin
Glycosaminoglycans

Keywords

  • antithrombins
  • binding sites
  • binding, competitive
  • electrophoretic mobility shift assay
  • heparin
  • hirudins
  • humans
  • hydrophobic and hydrophilic interactions
  • kinetics
  • models, molecular
  • mutagenesis, site-directed
  • mutation
  • osmolar concentration
  • peptide fragments
  • polyphosphates
  • protein binding
  • protein conformation
  • prothrombin
  • sodium chloride
  • surface plasmon resonance
  • thrombin

Cite this

Polyphosphate binds with high affinity to exosite II of thrombin. / Mutch, N J; Myles, T; Leung, L L K; Morrissey, J H.

In: Journal of Thrombosis and Haemostasis, Vol. 8, No. 3, 03.2010, p. 548-555.

Research output: Contribution to journalArticle

Mutch, N J ; Myles, T ; Leung, L L K ; Morrissey, J H. / Polyphosphate binds with high affinity to exosite II of thrombin. In: Journal of Thrombosis and Haemostasis. 2010 ; Vol. 8, No. 3. pp. 548-555.
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abstract = "Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C-terminal dodecapeptide and γ-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na+-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (Kd approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nmKd for the polyphosphate–thrombin interaction.",
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T1 - Polyphosphate binds with high affinity to exosite II of thrombin

AU - Mutch, N J

AU - Myles, T

AU - Leung, L L K

AU - Morrissey, J H

PY - 2010/3

Y1 - 2010/3

N2 - Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C-terminal dodecapeptide and γ-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na+-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (Kd approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nmKd for the polyphosphate–thrombin interaction.

AB - Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C-terminal dodecapeptide and γ-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na+-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (Kd approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nmKd for the polyphosphate–thrombin interaction.

KW - antithrombins

KW - binding sites

KW - binding, competitive

KW - electrophoretic mobility shift assay

KW - heparin

KW - hirudins

KW - humans

KW - hydrophobic and hydrophilic interactions

KW - kinetics

KW - models, molecular

KW - mutagenesis, site-directed

KW - mutation

KW - osmolar concentration

KW - peptide fragments

KW - polyphosphates

KW - protein binding

KW - protein conformation

KW - prothrombin

KW - sodium chloride

KW - surface plasmon resonance

KW - thrombin

U2 - 10.1111/j.1538-7836.2009.03723.x

DO - 10.1111/j.1538-7836.2009.03723.x

M3 - Article

C2 - 20002544

VL - 8

SP - 548

EP - 555

JO - Journal of Thrombosis and Haemostasis

JF - Journal of Thrombosis and Haemostasis

SN - 1538-7933

IS - 3

ER -