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

我们建立了一种酶免疫测定法，用于测定纤溶参数，如组织型纤溶酶原激活剂(t-PA)和尿激活酶(UK)。将硅胶片与第一抗体偶联，与样品孵育。孵育后，将硅胶片洗涤，然后与含有标记第二抗体的β-D-半乳糖苷酶的溶液孵育。测定β-D-半乳糖苷酶活性。以抗PAI-1的单抗为第一抗体，以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PAI-1、游离PAI-1和t-PA-PAI-1复合体浓度的检测系统。测定血浆中t-PA-PAI-1复合体的含量，用t-PA预孵育的血浆测定总PAI-1的含量，从总PAI-1中减去t-PA-PAI-1复合体的含量得到游离PAI-1的含量。血浆t-PA和t-PA-PAI-1复合体水平随年龄增长而升高。血浆t-PA水平男性高于女性，女性随着年龄的增长逐渐升高，达到与男性60岁时相同的水平。男性血浆t-PA-PAI-1复合体和游离PAI-1水平也高于女性。测定纤溶昼夜节律(8：30、10：30、12：30、14：30、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，雌性ELT始终短于雄性，t-PA和游离PAI-1水平早晨最高，晚上最低。结果表明，男性血浆纤溶参数的平衡水平高于女性，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.