Department of Translational Research, Stem Cells Unit, Centro di Riferimento Oncologico-IRCCS, National Cancer Institute, Aviano, Italy;
Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy; and.
Blood. 2015 Jan 22;125(4):697-705. doi: 10.1182/blood-2014-06-579474. Epub 2014 Dec 5.
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.
一氧化氮（NO）具有血管扩张、抗血小板、抗氧化和抗增殖作用。内皮细胞衍生的 NO 对心血管保护至关重要，而血小板合成并调节血小板功能的证据仍然存在争议。我们使用一种灵敏和特异的荧光探针，4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯（DAF-FM），在流动条件下观察到单个血小板在胶原底物上黏附时的 NO 产生。实时监测的 NO 产生依赖于施加的剪切率，随着剪切率的升高而增加。此外，在存在 l-精氨酸（一氧化氮合酶 [NOS] 底物）的情况下，NO 产生增加，而在存在 L-NG-单甲基精氨酸（NOS 抑制剂）的情况下，NO 产生减少，但在存在 D-NG-单甲基精氨酸（L-NMMA 非活性对映体）的情况下不减少。用 mepacrine 标记的血小板测量的血小板沉积与 NO 产生呈反比关系。Ca(++) 升高与 NO 产生之间存在明显的相关性，表明血小板 NO 的形成是由细胞内 Ca(++) 升高引发的。同时测量 NO 和 Ca(++) 表明，个体血小板中的 NO 产生先于 Ca(++) 升高，滞后期为 33 ± 9.5 s。我们的研究首次直接证明了在流动条件下，与激活表面相互作用可触发血小板产生 NO，并表明血小板内 Ca(++) 升高引发 NO 的产生，而 NO 反过来又调节血栓形成的大小。
