ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets

Duen Suey Chou, George Hsiao, Ming Yi Shen, Yan Jyu Tsai, Tzeng-Fu Chen, Joen Rong Sheu

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and α-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.

Original languageEnglish
Pages (from-to)237-248
Number of pages12
JournalFree Radical Biology and Medicine
Volume39
Issue number2
DOIs
Publication statusPublished - Jul 15 2005

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Spin Trapping
Electron Spin Resonance Spectroscopy
Prostaglandin-Endoperoxide Synthases
Platelets
Free Radicals
Paramagnetic resonance
Blood Platelets
Carbon
Arachidonic Acid
Peroxides
Platelet Aggregation
Glutathione
Potassium Cyanide
Agglomeration
Pronase
Deferoxamine
Thrombin
Biosynthesis
Prostaglandins
Digestion

Keywords

  • Carbon-centered free radical
  • Electron spin resonance
  • Peroxides
  • Platelets
  • Prostaglandin H synthase

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Cite this

ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets. / Chou, Duen Suey; Hsiao, George; Shen, Ming Yi; Tsai, Yan Jyu; Chen, Tzeng-Fu; Sheu, Joen Rong.

In: Free Radical Biology and Medicine, Vol. 39, No. 2, 15.07.2005, p. 237-248.

Research output: Contribution to journalArticle

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AB - Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and α-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.

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