Evidence for S-nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation
Evidence for S-nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation
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DOI：
10.1073/pnas.142136499
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发表时间：
2002-07-09
影响因子：
11.1
通讯作者：
Mutus, B
中科院分区：
文献类型：
--
作者：
Akhter, S;Vignini, A;Mutus, B
s -亚硝基谷胱甘肽（GSNO, 50 muM）抑制凝血酶催化的人和牛纤维蛋白原聚合的初始速率分别约为50%至68%。其他结构不同的s -亚硝基硫醇（RSNOs）也有抑制作用，包括s -亚硝基-n -乙酰青霉胺（SNAP）的糖衍生物。相同浓度的GSNO对凝血酶依赖性水解tosylglycyylprolylarginin -4-nitroanilide acetate没有影响，这表明这种抑制是由于GSNO诱导纤维蛋白原结构的改变。这一结果被CID光谱证实，GSNO或s -亚硝基同型半胱氨酸分别使纤维蛋白原的a-螺旋含量增加了约15%和11%。s -羧甲基酰胺衍生物谷胱甘肽或同型半胱氨酸对纤维蛋白原二级结构没有影响。在凝胶过滤层析上，gsno依赖性的二级结构效应被逆转，表明这种效应是变构的。纤维蛋白原-GSNO相互作用的进一步证据来自于GSNO依赖的内在纤维蛋白原色氨酸荧光猝灭和GSNO圆二色性吸光度作为[纤维蛋白原]的函数的扰动。等温滴定量热法和荧光猝灭法分别估算了纤维蛋白原-GSNO在化学计量比为2:1 （GSNO：纤维蛋白原）时3 ~ 10 ma的K(d)s。这些结果表明，RSNOs通过在特定的富芳香结构域相互作用诱导纤维蛋白原结构的变化。在纤维蛋白原α链的无序、芳香残基丰富的c端，已经发现了三个这样的假定的rsno结合结构域。
S-nitrosoglutathione (GSNO, 50 muM) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by approximate to50% to 68% respectively. inhibition was also observed with other structurally varied S-nitrosothiols (RSNOs) including sugar derivatives of S-nitroso-N-acetylpenicillamine (SNAP). The fact that the same concentration of GSNO had no effect on thrombin-dependent hydrolysis of tosylglycylprolylarginine-4-nitroanilide acetate suggested that this inhibition was due to GSNO-induced changes in fibrinogen structure. This result was confirmed by CID spectroscopy where GSNO or S-nitrosohomocysteine increased the a-helical content of fibrinogen by approximate to15% and 11 %, respectively. S-carboxymethylamido derivatives of glutathione or homocysteine had no effect on the fibrinogen secondary structure. The GSNO-dependent secondary structural effects were reversed on gel filtration chromatography, suggesting that the effects were allosteric. Further evidence for fibrinogen-GSNO interactions was obtained from GSNO-dependent quenching of the intrinsic fibrinogen Trp fluorescence and the perturbation of the GSNO circular dichroic absorbance as a function of [fibrinogen]. The K(d)s of 3 to 10 muM for fibrinogen-GSNO interactions with a stoichiometry of 2:1 (GSNO:fibrinogen) were estimated from isothermal titration calorimetry and fluorescence quenching, respectively. These results suggest that RSNOs induce changes to fibrinogen structure by interacting at specific aromatic rich domains. Three such putative RSNO-binding domains have been identified in the unordered, aromatic residue-rich C-termini of the alpha-chains of fibrinogen.