Freedman J E, Loscalzo J, Barnard M R, Alpert C, Keaney J F, Michelson A D
Whitaker Cardiovascular Institute and Evans Memorial Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Clin Invest. 1997 Jul 15;100(2):350-6. doi: 10.1172/JCI119540.
Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.
血管损伤和血栓形成是大多数缺血性冠状动脉综合征的病因，在此情况下，活化的血小板会刺激血小板聚集到正在生长的血栓处。最近，在人类血小板中发现了一种组成型一氧化氮合酶（NOS）。为了进一步确定血小板产生一氧化氮（NO）的能力，以及研究这种NO在血小板募集中的作用，我们对一种NO选择性微电极进行了改造，使其能用于标准血小板聚集仪，从而可以同时测量血小板聚集和NO产生。用NO合酶抑制剂-NG-硝基-L-精氨酸甲酯（L-NAME）处理血小板，与对照组相比，在5 microM ADP刺激下，NO产生减少了92±8%，但聚集仅增加了15±2%。相比之下，L-NAME对血小板募集有更显著的影响，表现为聚集程度增加35±5%，环磷酸鸟苷（cGMP）含量降低33±3%，以及在NO产生峰值时添加到受刺激血小板中的第二批可募集血小板中5-羟色胺释放增加31±5%。为了准确研究血小板募集，我们开发了一种能同时监测两个血小板群体的检测方法。将未生物素化的血小板与L-NAME或溶剂孵育，并用ADP激活。在NO产生峰值时，加入生物素化的血小板。通过三色流式细胞术测量，与对照组相比，用L-NAME处理的未生物素化群体中P选择素阳性血小板数量增加了56±11%。当将生物素化血小板添加到用L-NAME处理的未生物素化群体中时，与添加到对照组的生物素化血小板相比，P选择素阳性生物素化血小板数量增加了180±32%。总之，受刺激的血小板产生的NO适度抑制血小板活化，但显著抑制额外的血小板募集。这些数据表明，血小板衍生的NO可能调节血小板向正在生长的血栓的募集。
