Gombau L, Schleef R R
Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037.
J Biol Chem. 1994 Feb 4;269(5):3875-80.
To understand the processing of type 1 plasminogen activator inhibitor (PAI-1) into the storage granules of platelets, we utilized a eukaryotic expression vector (pRC/CMV) to transfer the human cDNA for PAI-1 into AtT-20 cells, a mouse pituitary cell line known to sort proteins in a regulated fashion. Immunofluorescence staining of PAI-1-transfected AtT-20 clones revealed co-localization of PAI-1 with an endogenously produced and stored hormone (i.e. adrenocorticotropic hormone, ACTH). Stimulation of PAI-1-transfected AtT-20 cells with a secretagogue resulted in the release of both active PAI-1 and the latent form. In comparison, PAI-1-transfected Chinese hamster ovary cells (i.e. a nonpackaging cell line) did not release PAI-1 in response to a secretagogue and exhibited immunoreactivity for PAI-1 primarily confined to the Golgi region. Percoll density gradient fractionation of AtT-20 cells revealed a codistribution of PAI-1 and ACTH in cellular compartments of the same density. The half-life of PAI-1 activity at 37 degrees C was prolonged in intact granules (t1/2, 5 h) in comparison with its half-life in lysed granules (t1/2, 2 h). These studies demonstrate the presence of a new functional property associated with the PAI-1 molecule that directs this inhibitor into the storage secretory pathway.
为了解1型纤溶酶原激活物抑制剂（PAI-1）进入血小板储存颗粒的过程，我们利用真核表达载体（pRC/CMV）将人PAI-1的cDNA导入AtT-20细胞，这是一种已知能以调节方式分选蛋白质的小鼠垂体细胞系。对转染PAI-1的AtT-20克隆进行免疫荧光染色，结果显示PAI-1与内源性产生并储存的激素（即促肾上腺皮质激素，ACTH）共定位。用促分泌剂刺激转染PAI-1的AtT-20细胞，可导致活性PAI-1和潜伏形式的PAI-1都释放出来。相比之下，转染PAI-1的中国仓鼠卵巢细胞（即非包装细胞系）在受到促分泌剂刺激时不释放PAI-1，其PAI-1免疫反应性主要局限于高尔基体区域。对AtT-20细胞进行Percoll密度梯度分级分离，结果显示PAI-1和ACTH在相同密度的细胞区室中共分布。与溶解颗粒中PAI-1活性的半衰期（t1/2，2小时）相比，完整颗粒中PAI-1活性在37℃时的半衰期延长（t1/2，5小时）。这些研究表明，PAI-1分子具有一种新的功能特性，可将这种抑制剂导向储存分泌途径。
