Alpha2-plasmin inhibitor and alpha2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibody immunosorbent assay.
Alpha2-plasmin inhibitor and alpha2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibody immunosorbent assay.
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血浆中的 α2-纤溶酶抑制剂和 α2-巨球蛋白-纤溶酶复合物。
DOI：
10.1172/jci110253
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Harpel,PC
中科院分区：
文献类型：
--
作者：
Harpel,PC
已开发了用于定量α 2-纤溶酶前体-纤溶酶和α 2-巨球蛋白-纤溶酶复合物的酶联差异抗体免疫吸附测定。在该方法中，通过一种底物特异性抗体将底物-纤溶酶复合物结合到一个表面上，并通过第二种抗体，即用碱性磷酸酶标记的兔抗纤溶酶原F（ab '）2，对结合到抑制剂上的纤溶酶进行定量。碱性磷酸酶对磷酸对硝基苯酯的水解以每毫升纤溶酶原的飞摩尔表示，参照标准纤溶酶原曲线。通过加入纤溶酶或尿激酶在血浆中产生抑制剂-酶复合物。加入的纤溶酶的浓度远低于α 2-纤溶酶抑制剂（1 μ M）或α 2-巨球蛋白（3.5 μ M）的血浆浓度，使得两种抑制剂都不会被酶完全饱和。在这些条件下，增加纤溶酶的量产生α 2-纤溶酶前体-纤溶酶和α 2-巨球蛋白-纤溶酶复合物的增加。将不同量的纤溶酶与血浆中发现的浓度的每种纯化的抑制剂和复合物一起孵育。将不同量的纤溶酶与血浆中发现的浓度的每种纯化的抑制剂一起孵育，并通过免疫测定定量形成的复合物。这些研究使得有可能量化纤溶酶或尿激酶处理的血浆中两种抑制剂之间的纤溶酶分布。在纤溶酶处理的血浆中，10%或更少的纤溶酶结合两种抑制剂与α 2-巨球蛋白复合。相比之下，尿激酶激活血浆中产生的纤溶酶中有19 - 51%与α 2-巨球蛋白结合。因此，根据是否向血浆中加入纤溶酶或纤溶酶原是否被内源性激活，观察到血浆分布的主要变化。尿激酶治疗性输注在体内产生的抑制剂纤溶酶复合物的模式与尿激酶活化血浆中发现的相似。23名正常人有低水平的α 2-纤溶酶前体-纤溶酶复合物，而6名实验室证据显示弥散性血管内凝血的患者显示这些复合物的浓度增加了16- 35倍。这些结果表明，该探针为纤溶酶系统的研究提供了一种新的探针。
An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by the therapeutic infusion of urokinase was similar to that found for urokinase-activated plasma. 23 normal individuals had low levels of alpha2-plasmin inhibitor-plasmin complexes, whereas six patients with laboratory evidence for disseminated intravascular coagulation demonstrated a 16- to 35-fold increase in he concentration of these complexes. These data indicated that a useful new probe for the study of the fibrinolytic enzyme system had been developed.