Visualization of nitric oxide production by individual platelets during adhesion in flowing blood.

Abstract:

:Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of l-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca(++) elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca(++) elevation. Simultaneous measurement of NO and Ca(++) indicated that NO production in individual platelets is preceded by Ca(++) elevations, with a lag phase of 33 ± 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca(++) elevation elicits the production of NO which, in turn, modulates thrombus size.

journal_name

Blood

journal_title

Blood

authors

Cozzi MR,Guglielmini G,Battiston M,Momi S,Lombardi E,Miller EC,De Zanet D,Mazzucato M,Gresele P,De Marco L

doi

10.1182/blood-2014-06-579474

subject

Has Abstract

pub_date

2015-01-22 00:00:00

pages

697-705

issue

4

eissn

0006-4971

issn

1528-0020

pii

blood-2014-06-579474

journal_volume

125

pub_type

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