Binding of tissue plasminogen activator to human aortic endothelial cells.
The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.
Sanzo
M A
MA
Department of Cell Culture and Biochemistry, Monsanto Company, St. Louis, MO 63167.
Howard
S C
SC
Wittwer
A J
AJ
Cochrane
H M
HM
D001011:Aorta; D001665:Binding Sites; D002460:Cell Line; D003106:Colon / Q000032:analysis; D004730:Endothelium, Vascular / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008545:Melanoma / Q000032:analysis; D015394:Molecular Structure; D015849:Plasminogen Inactivators / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator