title: Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation.
abstract: Ternary complex formation of tissue plasminogen activator (TPA) and plasminogen (Plg) with thrombospondin (TSP) or histidine-rich glycoprotein (HRGP) has been demonstrated using an enzyme-linked immunosorbent assay, an affinity bead assay, and a rocket immunoelectrophoresis assay. The formation of these complexes was specific, concentration dependent, saturable, lysine binding site-dependent, and inhibitable by fluid phase plasminogen. Apparent Kd values were approximately 12-36 nM for the interaction of TPA with TSP-Plg complexes and 15-31 nM with HRGP-Plg complexes. At saturation the relative molar stoichiometry of Plg:TPA was 3:1 within the TSP-containing complexes and 1:1 within HRGP-containing complexes. The activation of Plg to plasmin by TPA on TSP- and HRGP-coated surfaces was studied using a synthetic fluorometric plasmin substrate (D-Val-Leu-Lys-7-amino-4-trifluoromethyl coumarin). Kinetic analysis demonstrated a marked increase in the affinity of TPA for plasminogen in the presence of surface-associated TSP or HRGP. Compared to fluid phase activation or activation on fibronectin- or Factor VIII-related antigen-coated surfaces there was a 35-fold increase in efficiency of plasmin generation. A substantial amount (up to 71%) of the plasmin formed remained surface-associated and was found to be protected from inhibition by alpha 2-plasmin inhibitor. Greater than 200-fold increase in inhibitor concentration was required to effect 50% inhibition. Complex formation of locally released tissue plasminogen activator with Plg immobilized on TSP or HRGP surfaces may thus play an important role in effecting proteolytic events in nonfibrin-containing microenvironments.
authors.forename: R L
authors.initials: RL
authors.lastname: Silverstein
authors.forename: R L
authors.initials: RL
authors.lastname: Nachman
authors.forename: L L
authors.initials: LL
authors.lastname: Leung
authors.forename: P C
authors.initials: PC
authors.lastname: Harpel
mesh_terms: D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D010958:Plasminogen / Q000302:isolation & purification / Q000378:metabolism*; D010960:Plasminogen Activators / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin