Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator.
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.
Bartha
K
K
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Declerck
P J
PJ
Moreau
H
H
Nelles
L
L
Collen
D
D
D000941:Antigens / Q000235:genetics; D001665:Binding Sites; D015152:Blotting, Northern; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D015849:Plasminogen Inactivators / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics*
D000941:Antigens; D015849:Plasminogen Inactivators; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator