Laschinger C A, Johnston M G, Hay J B, Wasi S
Protein Chemistry Section, National Reference Laboratory, Canadian Red Cross Society, Ottawa, Ontario.
Thromb Res. 1990 Aug 1;59(3):567-79. doi: 10.1016/0049-3848(90)90416-a.
We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.
我们研究了培养的淋巴管内皮细胞是否产生纤溶酶原激活剂（PAs）及其抑制剂（PAIs），以及这些活性是否可被炎性细胞因子肿瘤坏死因子α（TNF-α）调节。通过反向纤维蛋白自显影检查这些细胞的条件培养基，发现了一种分子量为50 kDa的PAI。纤维蛋白自显影还显示出两种PA，分子量分别为110 kDa和60 kDa。110 kDa的蛋白与PA-PAI复合物共迁移，60 kDa的蛋白与组织纤溶酶原激活剂（tPA）共迁移。功能和免疫分析表明，人TNF-α以时间依赖性方式增加1型纤溶酶原激活剂抑制剂（PAI-1）。用重组人TNF-α处理细胞24小时后，释放到条件培养基中的PAI量增加了3至7倍。免疫印迹分析确定TNF-α处理的细胞条件培养基中的PAI为PAI-1。然后PAI-1在细胞外基质中的沉积变得明显。TNF-α使条件培养基中检测到的tPA-PAI-1复合物（分子量110 kDa）的量增加了4倍。游离tPA（分子量60 kDa）降至对照的1/5。用发色底物测定法测定，TNF-α处理后净纤溶活性降低。在对照或处理的细胞中未检测到尿激酶型纤溶酶原激活剂（uPA）活性。这种纤溶活性对于维持间质和淋巴管中的自由液体流动可能很重要，在炎症状态下，PAI-1的增加可能会降低这种潜力。
