Tannous M, Rabini R A, Vignini A, Moretti N, Fumelli P, Zielinski B, Mazzanti L, Mutus B
School of Physical Sciences, Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Diabetologia. 1999 May;42(5):539-44. doi: 10.1007/s001250051192.
AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase.
Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting.
In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase.
CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.
目的/假设：本研究有两个目的。其一，确定糖尿病患者的血小板是否比正常血小板产生更多的过氧亚硝酸盐；其二，将过氧亚硝酸盐的产生与血小板内诱导型一氧化氮合酶的诱导情况相关联。
采用二氯荧光素乙酸酯结合共聚焦显微镜和稳态荧光监测血小板内过氧亚硝酸盐的产生。通过蛋白质免疫印迹法检测血小板中诱导型一氧化氮合酶的诱导情况。
在细胞外L-精氨酸（100微摩尔/升）存在的情况下，I型（胰岛素依赖型）糖尿病患者的血小板荧光比对照组高约5倍。为了确定诱导型一氧化氮合酶是否是过氧亚硝酸盐的来源，在对照组、I型糖尿病患者和II型（非胰岛素依赖型）糖尿病患者的血小板中，将二氯荧光素的产生量作为L-精氨酸和一氧化氮合酶抑制剂的函数进行定量。I型糖尿病患者的血小板产生的L-精氨酸/一氧化氮合酶依赖性二氯荧光素荧光量比对照组高约7倍，II型糖尿病患者的血小板则高约3倍。然后对血小板进行免疫检测以寻找诱导型一氧化氮合酶，此前已发现其与过氧亚硝酸盐的产生有关，并在冠心病患者的巨核细胞中检测到。血小板内蛋白质的蛋白质免疫印迹表明，对照组中不存在诱导型一氧化氮合酶。然而，I型和II型糖尿病患者的血小板中均含有诱导型一氧化氮合酶。
结论/解读：诱导型一氧化氮合酶衍生的过氧亚硝酸盐是糖尿病中血小板损伤的一个来源。
