Reversible Activation of Cellular Factor XIII by Calcium
Reversible Activation of Cellular Factor XIII by Calcium
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DOI：
10.1074/jbc.m110.174128
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发表时间：
2011-03-18
影响因子：
4.8
通讯作者：
Andersen, Mette Dahl
中科院分区：
文献类型：
--
作者：
Kristiansen, Gunhild Klarskov;Andersen, Mette Dahl
XIII 因子 (FXIII) 是一种转谷氨酰胺酶原，存在于血浆以及细胞内的血小板和巨噬细胞中。血浆 FXIII 由凝血酶裂解 (FXIIIa*) 激活，并在凝血级联的最后阶段发挥作用。相比之下，细胞 FXIII 的功能和激活特征较少。细胞 FXIII 依赖于蛋白质的构象激活。仅由 Ca2+ 诱导的 FXIII 至 FXIIIa 程度的非蛋白水解激活是众所周知的，但到目前为止，人们一直在讨论在什么条件下可以诱导该过程以及是否可以逆转该过程。在这里，我们研究 Ca2+ 诱导的 FXIII 激活的性质。以前用于评估 FXIII 活性的方法可检测 FXIIIa* 和 FXIIIa 程度，因为它们依赖于酶活性的发生或活性位点 Cys-314 溶剂可及性。因此，开发了一种分析 HPLC 方法，将酶原重组 FXIII (rFXIII) 与 rFXIIIa 程度分开。数据表明，非蛋白水解激活和失活高度依赖于 Ca2+ 浓度、缓冲液和盐成分。此外，已确定 rFXIII 的 Ca2+ 激活是完全可逆的，仅 2-5 mM CaCl2 就足以保留完整的 rFXIIIa 度活性。然而，低于 2 mM CaCl2，rFXIIIa 度分子会失活。失活的分子随后可以经历新一轮的激活。此外，研究表明，冻干 rFXIII 的热应激可以诱导一种新的易感形式，其激活速度比未应激的 rFXIII 更快。
Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa degrees induced by Ca2+ alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca2+ -induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa degrees because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa degrees. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca2+ concentration, buffer, and salt components. Moreover, it is established that Ca2+ activation of rFXIII is fully reversible, and only 2-5 mM CaCl2 is sufficient to retain full rFXIIIa degrees activity. However, below 2 mM CaCl2 the rFXIIIa degrees molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.