Bartha K, Declerck P J, Moreau H, Nelles L, Collen D
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
J Biol Chem. 1991 Jan 15;266(2):792-7.
The effects of recombinant tissue-type plasminogen activator (rt-PA) and of an inactive mutant of rt-PA, obtained by mutagenesis of the active site Ser478 to Ala (rt-PA-Ala478), on the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture were studied. Under base-line conditions, PAI-1 antigen secretion was 4.3 +/- 1.0 micrograms (mean +/- S.D., n = 8) per 10(6) cells in 24 h. This PAI-1 had a low specific activity (6,000 +/- 1,600 units/mg) and Mr of 50,000, which was not altered by addition of rt-PA. In HUVEC cultured with 2 micrograms/ml rt-PA-Ala478, PAI-1 antigen secretion was 2.1 +/- 0.8 micrograms (n = 5) per 10(6) cells in 24 h with a specific activity of 120,000 +/- 42,000 units/mg and Mr of 50,000. Addition of rt-PA to this conditioned medium resulted in generation of three main components: 16% migrated as an Mr 106,000 rt-PA.PAI-1 complex, 16% as an Mr 81,000 degraded rt-PA.PAI-1 complex and the remainder as an Mr 45,000 degradation product of PAI-1. HUVEC cultured with 2 micrograms/ml rt-PA secreted 3.9 +/- 0.6 micrograms (n = 8) PAI-1 antigen per 10(6) cells within 24 h, of which 20-50% occurred as intact or degraded complexes with t-PA (Mr 106,000 and 81,000) and the rest as an inactive Mr 45,000 degradation product of PAI-1. PAI-1 mRNA levels, determined by Northern blot analysis and expressed relative to beta-actin mRNA levels, were very similar for HUVEC cultured in the absence or the presence of rt-PA or rt-PA-Ala478. It is concluded that PAI-1 is secreted by HUVEC in culture in fully active form which spontaneously inactivates. PAI-1 can be stabilized by addition of rt-PA-Ala478 to the culture medium, resulting in a 20-fold increase in specific activity. Interaction of rt-PA with active PAI-1 produces both t-PA.PAI-1 complex and an inactive degradation product of PAI-1.
研究了重组组织型纤溶酶原激活剂（rt-PA）及其通过将活性位点丝氨酸478突变为丙氨酸获得的无活性突变体（rt-PA-Ala478）对培养的人脐静脉内皮细胞（HUVEC）纤溶酶原激活剂抑制剂-1（PAI-1）合成和分泌的影响。在基线条件下，每10⁶个细胞在24小时内PAI-1抗原分泌量为4.3±1.0微克（平均值±标准差，n = 8）。这种PAI-1具有低比活性（6,000±1,600单位/毫克），分子量为50,000，添加rt-PA后未改变。在用2微克/毫升rt-PA-Ala478培养的HUVEC中，每10⁶个细胞在24小时内PAI-1抗原分泌量为2.1±0.8微克（n = 5），比活性为120,000±42,000单位/毫克，分子量为50,000。向这种条件培养基中添加rt-PA导致产生三种主要成分：16%迁移为分子量106,000的rt-PA.PAI-1复合物，16%为分子量81,000的降解rt-PA.PAI-1复合物，其余为分子量45,000的PAI-1降解产物。在用2微克/毫升rt-PA培养的HUVEC中，每10⁶个细胞在24小时内分泌3.9±0.6微克（n = 8）PAI-1抗原，其中20 - 50%以与t-PA的完整或降解复合物形式出现（分子量106,000和81,000），其余为无活性的分子量45,000的PAI-1降解产物。通过Northern印迹分析测定并相对于β-肌动蛋白mRNA水平表示的PAI-1 mRNA水平，对于在不存在或存在rt-PA或rt-PA-Ala478的情况下培养的HUVEC非常相似。得出的结论是，PAI-1由培养的HUVEC以完全活性形式分泌，其会自发失活。通过向培养基中添加rt-PA-Ala478可使PAI-1稳定，导致比活性增加20倍。rt-PA与活性PAI-1相互作用产生t-PA.PAI-1复合物和PAI-1的无活性降解产物。
