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Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation
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Human blood platelets lack nitric oxide synthase activity.

Anke Böhmer1, Stepan Gambaryan, Dimitrios Tsikas

  • 1Hannover Medical School, Institute of Clinical Pharmacology , Hannover , Germany .

Platelets
|November 1, 2014
PubMed
Summary

This study found no evidence of nitric oxide synthase (NOS) activity in human platelets using a sensitive GC-MS method. These findings challenge the presence of functional NOS in platelets and erythrocytes.

Keywords:
Enzyme activity assaysmass spectrometrynitric oxidestable isotopes

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Area of Science:

  • Biochemistry
  • Hematology
  • Physiology

Background:

  • Contradictory reports exist regarding nitric oxide synthase (NOS) expression and activity in human blood platelets and erythrocytes.
  • Previous research from our group has questioned the presence and function of NOS in these blood cells.

Purpose of the Study:

  • To definitively detect and quantify functional nitric oxide synthase (NOS) activity in human platelets.
  • To resolve discrepancies in the literature concerning NOS in platelets and erythrocytes.

Main Methods:

  • Utilized a specific gas chromatography-mass spectrometry (GC-MS) method to measure NOS activity.
  • Simultaneously detected [(15)N]nitrite and [(15)N]nitrate formed from oxidized (15)N-labeled nitric oxide ((15)NO).
  • Assayed NOS activity in non-stimulated, agonist-activated (ADP, collagen, thrombin, vWF), and lysed human platelets.

Main Results:

  • No functional NOS activity was detected in non-stimulated or activated human platelets, including lysed samples.
  • NOS activity was only measurable after the addition of recombinant human endothelial NOS to lysed platelets.
  • The specific inhibitor l-[guanidino-nitro]-Arginine blocked the added recombinant NOS activity.

Conclusions:

  • The study provides strong evidence against the presence of functional nitric oxide synthase (NOS) in human platelets.
  • Findings support previous work challenging the expression and functionality of NOS in human platelets and erythrocytes.
  • The employed GC-MS method offers high sensitivity for detecting NOS activity.