The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator.
The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.
Schleef
R R
RR
Research Institute of Scripps Clinic, Department of Immunology, La Jolla, California 92037.
Podor
T J
TJ
Dunne
E
E
Mimuro
J
J
Loskutoff
D J
DJ
D002455:Cell Division; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005342:Fibrinolysis; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008970:Molecular Weight; D015849:Plasminogen Inactivators / Q000032:analysis* / Q000378:metabolism; D011247:Pregnancy; D011863:Radioimmunoassay; D012996:Solutions; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014471:Umbilical Veins
D015849:Plasminogen Inactivators; D012996:Solutions; D010959:Tissue Plasminogen Activator