The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents

Isabella Russo, Gabriella Doronzo, Luigi Mattiello, Alessandro De Salve, Mariella Trovati, Giovanni Anfossi

Research output: Contribution to journalArticlepeer-review

Abstract

Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3′,5′- cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, β-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser 1177 and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of l-[ 3H]-arginine to l-[ 3H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 μmol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser 157 and Ser 239 and of ecNOS at Ser 1177 was evaluated by Western blot. NOS activity (pmol l-citrulline/10 8 platelets) increased from 0.090±0.002 to 0.148±0.013 with 500 μmol/l 8-Br-cAMP (p1177. The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.

Original languageEnglish
Pages (from-to)265-273
Number of pages9
JournalThrombosis Research
Volume114
Issue number4
DOIs
Publication statusPublished - 2004

Keywords

  • Adenosine
  • Cyclic AMP
  • Forskolin
  • Nitric oxide synthase
  • Platelets
  • Protein kinase A

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Hematology

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