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 thatNOis 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.
| Original language | English |
|---|---|
| Pages (from-to) | 697-705 |
| Number of pages | 9 |
| Journal | Blood |
| Volume | 125 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Jan 22 2015 |
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ASJC Scopus subject areas
- Hematology
- Biochemistry
- Cell Biology
- Immunology
- Medicine(all)
Cite this
Platelets and thrombopoiesis : Visualization of nitric oxide production by individual platelets during adhesion in flowing blood. / Cozzi, Maria Rita; Guglielmini, Giuseppe; Battiston, Monica; Momi, Stefania; Lombardi, Elisabetta; Miller, Edward Cole; De Zanet, Denise; Mazzucato, Mario; Gresele, Paolo; De Marco, Luigi.
In: Blood, Vol. 125, No. 4, 22.01.2015, p. 697-705.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Platelets and thrombopoiesis
T2 - Visualization of nitric oxide production by individual platelets during adhesion in flowing blood
AU - Cozzi, Maria Rita
AU - Guglielmini, Giuseppe
AU - Battiston, Monica
AU - Momi, Stefania
AU - Lombardi, Elisabetta
AU - Miller, Edward Cole
AU - De Zanet, Denise
AU - Mazzucato, Mario
AU - Gresele, Paolo
AU - De Marco, Luigi
PY - 2015/1/22
Y1 - 2015/1/22
N2 - 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 thatNOis 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.
AB - 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 thatNOis 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.
UR - http://www.scopus.com/inward/record.url?scp=84921629241&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84921629241&partnerID=8YFLogxK
U2 - 10.1182/blood-2014-06-579474
DO - 10.1182/blood-2014-06-579474
M3 - Article
C2 - 25480660
AN - SCOPUS:84921629241
VL - 125
SP - 697
EP - 705
JO - Blood
JF - Blood
SN - 0006-4971
IS - 4
ER -