Rouy D, Anglés-Cano E
Institut National de la Santé et de la Recherche Médicale, Hôpital de Bicêtre, France.
Biochem J. 1990 Oct 1;271(1):51-7. doi: 10.1042/bj2710051.
The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.
对在血浆环境或重组系统中，纤维蛋白结合的组织型纤溶酶原激活剂（t-PA）激活人谷氨酸纤溶酶原的机制进行了表征。使用了一种异质系统，该系统能够将实验条件设置得尽可能接近生理状态下的纤维蛋白/血浆界面，并允许根据时间对每个相中存在的产物进行单独分析。分别使用纤溶酶选择性显色底物和放射性标记的谷氨酸纤溶酶原，通过分光光度分析和放射性同位素分析来监测纤溶酶的生成。通过SDS/PAGE，然后进行放射自显影和/或免疫印迹来鉴定纤溶酶（原）衍生的产物。当在血浆环境中进行激活时，在纤维蛋白表面鉴定出的产物是谷氨酸纤溶酶（90%）和谷氨酸纤溶酶原（10%），而在可溶性相中仅检测到谷氨酸纤溶酶与其快速作用抑制剂之间的复合物。使用包含固相纤维蛋白、t-PA、谷氨酸纤溶酶原和α2-抗纤溶酶的重组系统也获得了相同的结果。相反，当溶液中省略α2-抗纤溶酶时，赖氨酸纤溶酶逐渐在纤维蛋白表面生成（30%）并释放到可溶性相中。在存在α2-抗纤溶酶或血浆的情况下，在纤维蛋白表面生成的活性纤溶酶量低于不存在抑制剂时的情况：在一个代表性实验中，对于200 nM纤溶酶原、200 nM纤溶酶原加100 nMα2-抗纤溶酶和天然血浆，纤溶酶生成的初始速度分别为2.8×10-3、2.0×10-3和1.8×10-3（ΔA405/min）。我们的结果表明，在血浆中或在含有与血浆中发现的比例相似的纤溶酶原和α2-抗纤溶酶的重组纯化系统中，（1）天然谷氨酸纤溶酶原的激活途径直接进行到谷氨酸纤溶酶的形成，（2）赖氨酸纤溶酶原不是反应的中间体，因此（3）赖氨酸纤溶酶不是最终的活性产物。然而，在不存在抑制剂的情况下，也会生成赖氨酸纤溶酶以及可能的赖氨酸纤溶酶原，赖氨酸纤溶酶原比谷氨酸纤溶酶原更容易被激活为纤溶酶。
