title: 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.
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Maria Rita
authors.initials: MR
authors.lastname: Cozzi
authors.affiliation: Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy; and.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Guglielmini
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Monica
authors.initials: M
authors.lastname: Battiston
authors.affiliation: Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy; and.
authors.forename: Stefania
authors.initials: S
authors.lastname: Momi
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Lombardi
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Edward Cole
authors.initials: EC
authors.lastname: Miller
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Denise
authors.initials: D
authors.lastname: De Zanet
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
authors.forename: Mario
authors.initials: M
authors.lastname: Mazzucato
authors.affiliation: Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy; and.
authors.forename: Paolo
authors.initials: P
authors.lastname: Gresele
authors.affiliation: Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.
authors.forename: Luigi
authors.initials: L
authors.lastname: De Marco
mesh_terms: D000818:Animals; D001783:Blood Flow Velocity; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000493:pharmacokinetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D010973:Platelet Adhesiveness / Q000187:drug effects / Q000502:physiology*; D011796:Quinacrine / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D003094:Collagen; D011796:Quinacrine; D002118:Calcium