title: Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand.
abstract: The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
authors.forename: X F
authors.initials: XF
authors.lastname: Cheng
authors.forename: O
authors.initials: O
authors.lastname: Bäck
authors.forename: T K
authors.initials: TK
authors.lastname: Nilsson
authors.forename: E
authors.initials: E
authors.lastname: Nylander Lundqvist
authors.forename: G
authors.initials: G
authors.lastname: Pohl
authors.forename: P
authors.initials: P
authors.lastname: Wallén
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D008024:Ligands; D046911:Macromolecular Substances; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000378:metabolism*; D014471:Umbilical Veins
chemical_list: D007457:Iodine Radioisotopes; D008024:Ligands; D046911:Macromolecular Substances; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010959:Tissue Plasminogen Activator