Department of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands.
Biochem Biophys Res Commun. 2012 Oct 19;427(2):421-5. doi: 10.1016/j.bbrc.2012.09.081. Epub 2012 Sep 21.
The ultimate step in the blood coagulation cascade is the formation of fibrin. Several proteins are known to bind to fibrin and may thereby change clot properties or clot function. Our previous studies identified carboxypeptidase N (CPN) as a novel plasma clot component. CPN cleaves C-terminal lysine and arginine residues from several proteins. The activity of CPN is increased upon its proteolysis by several proteases. The aim of this study is to investigate the presence of CPN in a plasma clot in more detail. Plasma clots were formed by adding thrombin, CaCl(2) and aprotinin to citrated plasma. Unbound proteins were washed away and non-covalently bound proteins were extracted and analyzed with 2D gel electrophoresis and mass spectrometry. The identification of CPN as a fibrin clot-bound protein was verified using Western blotting. Clot-bound CPN consisted of the same molecular forms as CPN in plasma and its content was approximately 30 ng/ml plasma clot. Using surface plasmon resonance we showed that CPN can bind to fibrinogen as well as to fibrin. In conclusion, CPN binds to fibrinogen and is present in a fibrin clot prepared from plasma. Because CPN binds to a fibrin clot, there could be a possible role for CPN as a fibrinolysis inhibitor.
凝血级联反应的最终步骤是纤维蛋白的形成。已知有几种蛋白质可以与纤维蛋白结合，从而改变血栓的性质或功能。我们之前的研究确定羧肽酶 N (CPN) 是一种新型的血浆血栓成分。CPN 从几种蛋白质中切割 C 末端赖氨酸和精氨酸残基。CPN 的活性在被几种蛋白酶水解后增加。本研究旨在更详细地研究 CPN 在血浆血栓中的存在情况。通过向柠檬酸盐血浆中加入凝血酶、CaCl2 和抑肽酶来形成血浆血栓。未结合的蛋白质被洗掉，非共价结合的蛋白质用 2D 凝胶电泳和质谱法进行分析。使用 Western blot 验证了 CPN 作为纤维蛋白血栓结合蛋白的存在。血栓结合的 CPN 与血浆中的 CPN 具有相同的分子形式，其含量约为 30ng/ml 血浆血栓。使用表面等离子体共振，我们表明 CPN 可以与纤维蛋白原以及纤维蛋白结合。总之，CPN 结合纤维蛋白原并存在于从血浆制备的纤维蛋白血栓中。由于 CPN 与纤维蛋白血栓结合，CPN 可能作为纤维蛋白溶解抑制剂发挥作用。
