Regulation of tissue plasminogen activator activity by cells. Domains responsible for binding and mechanism of stimulation.
A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.
Sinniger
V
V
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire EN6 3QG, United Kingdom.
Merton
R E
RE
Fabregas
P
P
Felez
J
J
Longstaff
C
C
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D008956:Models, Chemical; D011485:Protein Binding; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D017931:DNA Primers; D010959:Tissue Plasminogen Activator