Regulation of macrophage receptor-bound plasmin by autoproteolysis

• Published in: The Journal of biological chemistry Vol. 269; no. 51; pp. 32660 - 32666
• Main Authors: Falcone, D J, Borth, W, McCaffrey, T A, Mathew, J, McAdam, K
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 23-12-1994
• Subjects: alpha-2-Antiplasmin - metabolism; Catalysis; Cell Line; Cell Membrane - metabolism; Fibrinolysin - metabolism; Humans; Hydrolysis; Iodine Radioisotopes; Macrophages - metabolism; Phenylmethylsulfonyl Fluoride - metabolism; Plasminogen - metabolism; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; Urokinase-Type Plasminogen Activator - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)31685-5

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.