Catani M V, Bernassola F, Rossi A, Melino G
IDI-IRCCS, Biochemistry Laboratory, Department of Experimental Medicine, University of Rome Tor Vergata, Italy.
Biochem Biophys Res Commun. 1998 Aug 10;249(1):275-8. doi: 10.1006/bbrc.1998.9130.
The plasma factor XIII (FXIII) is a transglutaminase which catalyzes the cross-linking of fibrin monomers during blood coagulation. S-nitrosylation of protein sulfhydryl groups has been shown to regulate protein function. Therefore, to establish whether nitric oxide (NO) affects the enzymatic activity of FXIII, we studied the effect of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) in a blood coagulation test in vitro. High concentrations of SNAP were found to have inhibitory effects on clot formation. Moreover, specific formation of gamma-dimers through the action of FXIII is selectively inhibited by high concentrations of SNAP, as revealed by Western blot. Purified activated FXIII and plasma preparations were then exposed to NO-donor compounds and the enzyme activity was assayed by measuring the incorporation of [3H] putrescine into dimethylcasein. The NO donors, SNAP, spermine-NO (SPER-NO) and 3-morpholinosydnonimine (SIN-1), and the NO-carrier, S-nitrosoglutathione (GSNO), inhibited FXIII activity in a dose-dependent manner, in both purified enzyme and plasma preparations. Titration of -SH groups of FXIII with [14C] iodoacetamide has shown that the number of titratable cysteines per monomer of FXIII decreased from 1 (in absence of NO donors) to 0 (in the presence of NO donors). These results demonstrate that blood coagulation FXIII is a target for NO both in vitro and in vivo, and that inhibition occurs by S-nitrosylation of a highly reactive cysteine residue. In conclusion, we show that inhibition of FXIII activity by NO may represent an additional regulatory mechanism for the formation of blood clot with physio-pathological implications.
血浆因子 XIII（FXIII）是一种转谷氨酰胺酶，在血液凝固过程中催化纤维蛋白单体的交联。蛋白质巯基的 S-亚硝基化已被证明可调节蛋白质功能。因此，为了确定一氧化氮（NO）是否影响 FXIII 的酶活性，我们在体外血液凝固试验中研究了 NO 供体 S-亚硝基-N-乙酰青霉胺（SNAP）的作用。发现高浓度的 SNAP 对血凝块形成有抑制作用。此外，如 Western 印迹所示，高浓度的 SNAP 选择性抑制了通过 FXIII 作用形成的γ-二聚体。然后将纯化的活化 FXIII 和血浆制剂暴露于 NO 供体化合物，并通过测量 [3H] 腐胺掺入二甲基酪蛋白中来测定酶活性。NO 供体 SNAP、精胺-NO（SPER-NO）和 3-吗啉代-sydnonimine（SIN-1）以及 NO 载体 S-亚硝基谷胱甘肽（GSNO）在纯化酶和血浆制剂中均以剂量依赖性方式抑制 FXIII 活性。用 [14C] 碘乙酰胺滴定 FXIII 的 -SH 基团表明，FXIII 每个单体可滴定半胱氨酸的数量从 1（在无 NO 供体时）降至 0（在有 NO 供体时）。这些结果表明，血液凝固 FXIII 在体外和体内都是 NO 的作用靶点，并且抑制是通过高反应性半胱氨酸残基的 S-亚硝基化发生的。总之，我们表明 NO 对 FXIII 活性的抑制可能代表了一种对血凝块形成的额外调节机制，具有生理病理意义。
