Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties.
Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties.
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DOI：
10.1097/mbc.0000000000000602
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发表时间：
2017-07
期刊：
影响因子：
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通讯作者：
Kim-Shapiro DB
中科院分区：
文献类型：
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作者：
Helms CC;Kapadia S;Gilmore AC;Lu Z;Basu S;Kim-Shapiro DB
纤维蛋白纤维构成了血凝块的结构支柱。纤维蛋白凝块的结构特性高度依赖于形成动力学。环境因素，如蛋白质浓度、pH、盐和蛋白质修饰，仅举几例，可以通过改变纤维蛋白肽释放、单体结合和/或横向聚集来影响纤维动力学。测定凝血酶和纤维蛋白原暴露于一氧化氮对纤维蛋白凝块特性的影响。在凝块起始前和凝血酶加入凝血酶后立即加入5 μM的脯氨酸氨基酸（ProliNONOate）到纤维蛋白原和凝血酶溶液中。用原子力显微镜检测纤维蛋白纤维的直径和延展性，用共聚焦显微镜分析纤维蛋白凝块的密度。用共聚焦显微镜测定纤维直径，用紫外-可见分光光度法记录血块形成率。采用紫外-可见法、酶联免疫吸附法和化学发光法测定蛋白质氧化和s -亚硝化作用。在纤维蛋白原或凝血酶中加入脯氨酸酸盐可改变血块结构。脯氨酸酸盐暴露产生的凝块纤维密度较低，纤维较厚，达到最大浊度的时间延长。一氧化氮暴露于凝血酶和纤维蛋白原的影响是独立测量的，表明每一个都在改变凝块特性中起作用。我们检测了一氧化氮暴露后凝血酶s -亚硝化和蛋白质羰基形成。我们的研究揭示了一氧化氮暴露对纤维蛋白凝块特性的调节，并提示过氧亚硝酸盐在蛋白质氧化修饰中的作用。这些结果将一氧化氮的生物利用度和氧化应激与血栓特性的改变联系起来。
Fibrin fibers form the structural backbone of blood clots. The structural properties of fibrin clots are highly dependent on formation kinetics. Environmental factors such as protein concentration, pH, salt and protein modification, to name a few, can affect fiber kinetics through altered fibrinopeptide release, monomer association and/or lateral aggregation. To determine the effect of thrombin and fibrinogen exposed to nitric oxide on fibrin clot properties. ProliNONOate (5 μM) was added to fibrinogen and thrombin before clot initiation and immediately following the addition of thrombin to the fibrinogen solution. Resulting fibrin fibers were probed with an atomic force microscope to determine their diameter and extensibility and fibrin clots were analyzed for clot density using confocal microscopy. Fiber diameters were also determined by confocal microscopy and the rate of clot formation was recorded using UV-vis spectrophotometry. Protein oxidation and S-nitrosation was determined by UV-vis, ELISA and chemiluminescence. The addition of ProliNONOate to fibrinogen or thrombin resulted a change in clot structure. ProliNONOate exposure produced clots with lower fiber density, thicker fibers and increased time to maximum turbidity. The effect of the exposure of nitric oxide to thrombin and fibrinogen were measured independently and indicated that each plays a role in altering clot properties. We detected thrombin S-nitrosation and protein carbonyl formation after nitric oxide exposure. Our study reveals a regulation of fibrin clot properties by nitric oxide exposure and suggests a role of peroxynitrite in oxidative modifications of the proteins. These results relate NO bioavailability and oxidative stress to altered clot properties.