Akhter Shirin, Vignini Arianna, Wen Zhong, English Ann, Wang Peng G, Mutus Bulent
Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada N9B
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9172-7. doi: 10.1073/pnas.142136499. Epub 2002 Jun 27.
S-nitrosoglutathione (GSNO, 50 microM) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by approximately 50% 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 CD spectroscopy where GSNO or S-nitrosohomocysteine increased the alpha-helical content of fibrinogen by approximately 15% 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 microM 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.
S-亚硝基谷胱甘肽（GSNO，50微摩尔）分别使凝血酶催化的人纤维蛋白原和牛纤维蛋白原聚合反应的初始速率降低了约50%至68%。在其他结构各异的S-亚硝基硫醇（RSNOs）中也观察到了抑制作用，包括S-亚硝基-N-乙酰青霉胺（SNAP）的糖衍生物。相同浓度的GSNO对凝血酶依赖性的甲苯磺酰甘氨酰脯氨酰精氨酸-4-硝基苯胺醋酸盐的水解没有影响，这一事实表明这种抑制是由于GSNO诱导的纤维蛋白原结构变化所致。圆二色光谱证实了这一结果，其中GSNO或S-亚硝基高半胱氨酸分别使纤维蛋白原的α-螺旋含量增加了约15%和11%。谷胱甘肽或高半胱氨酸的S-羧甲基酰胺衍生物对纤维蛋白原的二级结构没有影响。GSNO依赖性的二级结构效应在凝胶过滤色谱上被逆转，这表明这些效应是别构效应。从GSNO依赖性猝灭纤维蛋白原的内在色氨酸荧光以及GSNO圆二色吸收随[纤维蛋白原]的变化中获得了纤维蛋白原与GSNO相互作用的进一步证据。通过等温滴定量热法和荧光猝灭分别估计了纤维蛋白原与GSNO相互作用的解离常数（K(d)）为3至10微摩尔，化学计量比为2:1（GSNO:纤维蛋白原）。这些结果表明，RSNOs通过在特定的富含芳香族结构域相互作用来诱导纤维蛋白原结构的变化。在纤维蛋白原α链无序的、富含芳香族残基的C末端已鉴定出三个这样的假定RSNO结合结构域。
