title: Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.
abstract: Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.
authors.affiliation: Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
authors.forename: V
authors.initials: V
authors.lastname: Ramakrishnan
authors.forename: D V
authors.initials: DV
authors.lastname: Sinicropi
authors.forename: R
authors.initials: R
authors.lastname: Dere
authors.forename: W C
authors.initials: WC
authors.lastname: Darbonne
authors.forename: K B
authors.initials: KB
authors.lastname: Bechtol
authors.forename: J B
authors.initials: JB
authors.lastname: Baker
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator