title: Regulation of macrophage receptor-bound plasmin by autoproteolysis.
abstract: The activation of plasminogen by macrophage is regulated by their expression of receptors for urokinase and plasmin(ogen). In these studies we have examined plasmin(ogen) binding to adherent human THP-1 macrophage. Plasmin bound to the THP-1 cells in a time- and dose-dependent manner (Kd 15.8 +/- 6.2 nM; Bmax 1.4 +/- 0.3 x 10(6)/cell). The lysine analog epsilon-aminocaproic acid competitively inhibited plasmin binding. The fraction of membrane-bound plasmin, however, became increasingly resistant to displacement with epsilon-aminocaproic acid. Over a 24-h period, membrane-bound plasmin activity fell 80% despite the presence of catalytically active plasmin in the incubation media. The loss of receptor-bound plasmin activity was not due to proteolytic alterations of its receptor since 125I-Lys-plasminogen bound to THP-1 cells pretreated with plasmin with similar affinity as to untreated cells. Following a 24-h incubation of 125I-Lys-plasminogen or 125I-plasmin with THP-1 cells, several degradative fragments were apparent in their conditioned media. The smaller degradative fragments (28 and 36 kDa) lacked cell binding activity and were demonstrated to be active by casein-zymography. A 48-kDa fragment bound to cells in a lysine-dependent manner but was not active. In contrast, phenylmethylsulfonyl fluoride-inactivated 125I-plasmin retained its binding activity over 24 h, and degradative fragments were not present in the conditioned media. The binding of 125I-Lys-plasmin(ogen) to THP-1 cells was also examined in the presence of excess alpha 2 plasmin inhibitor. Despite the absence of fluid-phase plasmin activity, membrane-bound 125I-Lys-plasmin(ogen) decreased over 24 h. At 24 h a radiolabeled 48-kDa fragment was observed in the conditioned media and together with 125I-Lys-plasmin(ogen) was bound to cells. Unlike 125I-Lys-plasmin, the 48-kDa fragment did not form a complex with alpha 2 plasmin inhibitor. Thus, autoproteolysis of receptor-bound plasmin results in fragments with truncated physiologic properties that possess either cell binding or catalytic activities. We propose that autoproteolysis is a mechanism for regulating membrane-bound plasmin activity.
authors.affiliation: Department of Pathology, Cornell Medical College, New York, New York.
authors.forename: D J
authors.initials: DJ
authors.lastname: Falcone
authors.forename: W
authors.initials: W
authors.lastname: Borth
authors.forename: T A
authors.initials: TA
authors.lastname: McCaffrey
authors.forename: J
authors.initials: J
authors.lastname: Mathew
authors.forename: K
authors.initials: K
authors.lastname: McAdam
mesh_terms: D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008264:Macrophages / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000378:metabolism
chemical_list: D007457:Iodine Radioisotopes; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D000979:alpha-2-Antiplasmin; D010664:Phenylmethylsulfonyl Fluoride; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator