Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells
Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells
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DOI：
10.1182/blood-2008-03-144279
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发表时间：
2009-01-08
期刊：
影响因子：
20.3
通讯作者：
Urano, Tetsumei
中科院分区：
文献类型：
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作者：
Suzuki, Yuko;Mogami, Hideo;Urano, Tetsumei
我们分析了电针中组织纤溶酶原激活物(TPA)的分泌动力学。Hy926细胞，一种已建立的产生GFP标记的tPA的血管内皮细胞系，使用全内反射-荧光(TIR-F)显微镜。电针小颗粒中可检测到tPAGFP。Hy926细胞，其分布与固有表达的tPA难以区分。它的分泌动力学是独特的，tPA-GFP在细胞表面长时间(5分钟)保留，在三分之二的胞吐事件中出现荧光点。TPA-GFP结构域缺失突变体的迅速消失(主要是250ms)表明tPAGFP的氨基末端重链是与膜表面结合所必需的。PAI-1的加入呈剂量依赖性地促进tPA的解离，并增加tPA-PAI-1高分子量复合体的数量。相应地，电针对PAI-1的合成有抑制作用。小干扰RNA延长了tPA-GFP的解离时间，而不与PAI-1形成复合物的tPA-GFP的催化失活突变体即使在PAI-1处理后仍保留在膜上。我们的结果为研究胞吐、膜滞留的tPA和PAI-1之间的关系提供了新的见解，PAI-1将调节细胞表面相关的纤溶潜力。(血。2009；113：470-478)
We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA. hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPAGFP was detected in small granules in EA. hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPAGFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecularweight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA. hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential. (Blood. 2009; 113: 470-478)