Mechanisms of the inhibition of t-PA by PAI-1

我们已经开发了一种酶免疫测定法，用于测量纤溶参数，如组织型纤溶酶原激活剂（t-PA）和尿激酶（UK）。将与第一抗体偶联的硅胶片与样品一起孵育。孵育后，洗涤硅胶片，然后与含有β-D-半乳糖苷酶标记的第二抗体的溶液一起孵育。测定β-D-半乳糖苷酶的活性。以抗派-1单克隆抗体为第一抗体，β-D-半乳糖苷酶标记的抗t-PA Fab'片段为第二抗体，建立了一种新的测定总派-1、游离派-1和t-PA-派-1复合物浓度的方法。从血浆中测量t-PA-派-1复合物的量，从用t-PA预孵育的血浆中测量总派-1的量，通过从总派-1的量中减去t-PA-派-1复合物的量获得游离派-1的量。血浆t-PA和t-PA-派-1复合物水平随年龄增长而升高。男性血浆t-PA水平高于女性，女性随年龄增长逐渐升高，60多岁时达到与男性相同的水平。男性t-PA-派-1复合物和游离派-1的血浆水平也高于女性。然后测定纤溶昼夜节律（8：30、10：30、12：30、14：30和16：30 h）。优球蛋白溶解时间（ELT）在8：30 ~ 10：30 h最长，16：30 h最短。女性ELT均短于男性，t-PA和游离派-1水平在早晨最高，晚上最低。这些结果表明，男性血浆中的纤溶参数平衡水平高于女性，但女性的纤溶活性高于男性。

We have developed an enzyme immunoassay for the measurements of fibrinolytic parameters such as tissue plasminogen activator (t-PA) and urokinase (UK). A silicone piece coupled with first antibody was incubated with a sample. After incubation the silicone piece was washed and then incubated with a solution containing beta-D-galactosidase labeled second antibody. The activity of beta-D-galactosidase was measured. Using a monoclonal antibody against PAI-1 as a first antibody and beta-D-galactosidase labeled anti- t-PA Fab' fragments as a second antibody, we developed a new assay system to measure the concentrations of total PAI-1, free PAI-1 and t-PA-PAI-1 complex. The amounts of t-PA-PAI-1 complex were measured from the plasma, and those of total PAI-1 were measured from plasma preincubated with t-PA, and those of free PAI-1 were obtained by subtractions of the amounts of t-PA-PAI-1 complex from those of total PAI-1. Plasma levels of t-PA and t-PA-PAI-1 complex increased with increase in age. Plasma levels of t-PA were higher in males than in females, and those in females gradually increased with increase in age, reaching the same levels as those of males at their 60s. Plasma levels of t-PA-PAI-1 complex and free PAI-1 were also higher in males than in females. Then circadian rhythm (8:30, 10:30, 12:30, 14:30 and 16:30 h) of fibrinolysis was measured. Euglobulin lysis time (ELT) showed longest time at 8:30 to 10:30 h and shortest time at 16:30 h. ELT was always shorter in females than in males, The highest level of t-PA or free PAI-1 was obtained in the morning and the lowest was in the evening. These results indicate that the fibrinolytic parameters were balanced at higher levels in plasma in males than in females, but the fibrinolytic activity was higher in females than in males.