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 plasminoge n activator (tPA), which in some cases can remain active on the cell surfac e resulting in enhanced plasminogen activation kinetics. We have investigat ed several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and show n 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 kin etic assays with a range of tPA domain deletion mutants consisting of full- length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), Delta FtPA (G-K1 -K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P), Deletio n variants were made in Escherichia coli and were nonglycosylated, Plasmino gen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum le vels of stimulation up to 80-fold with full-length, glycosylated tPA, Stimu lation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in Delta FtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The pr otease domain alone was stimulated at very low levels of up to a-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 tP A activity by fibrin, A model was developed to explain the mechanism of sti mulation and compared with actual data collected with varying cell, plasmin ogen, or tPA concentrations and different tPA variants. Experimental data a nd model predictions were generally in good agreement and suggest that stim ulation is well explained by the concentration of reactants by cells.