Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA.
Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.
Fujii
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S
Cardiovascular Division, Washington University School of Medicine, St. Louis, Missouri 63110.
Sobel
B E
BE
D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D002478:Cells, Cultured; D003470:Culture Media; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011233:Precipitin Tests; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000502:physiology*
D003470:Culture Media; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator