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Regulation of macrophage receptor-bound plasmin by autoproteolysis
D J Falcone1, W Borth, T A McCaffrey
1Department of Pathology, Cornell Medical College, New York, New York.
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.
Insights
Macrophage receptors bind plasmin, but its activity decreases over time due to autoproteolysis. This process generates fragments with altered cell-binding and catalytic properties, regulating membrane-bound plasmin activity.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Macrophage-mediated plasminogen activation is crucial for various physiological processes.
- Receptors for urokinase and plasmin(ogen) on macrophages regulate this activation.
- Understanding plasmin binding and activity on macrophages is key to deciphering inflammatory and tissue remodeling roles.
Purpose of the Study:
- To investigate the binding characteristics of plasmin to human THP-1 macrophages.
- To determine the stability and activity of receptor-bound plasmin over time.
- To identify mechanisms regulating membrane-bound plasmin activity on macrophages.
Main Methods:
- Quantifying plasmin binding to THP-1 cells using radiolabeled ligands (125I-Lys-plasminogen, 125I-plasmin).
- Assessing the effect of lysine analogs (epsilon-aminocaproic acid) and inhibitors (phenylmethylsulfonyl fluoride, alpha 2 plasmin inhibitor) on binding and activity.
- Analyzing plasmin degradation products in conditioned media using zymography and SDS-PAGE.
Main Results:
- Plasmin binds to THP-1 cells with high affinity (Kd 15.8 nM) in a time- and dose-dependent manner.
- Membrane-bound plasmin activity significantly decreases (80% over 24h) due to autoproteolysis, not receptor degradation.
- Autoproteolysis generates fragments with distinct properties: smaller fragments (28, 36 kDa) are active but do not bind cells, while a 48 kDa fragment binds cells but lacks activity.
Conclusions:
- Autoproteolysis of receptor-bound plasmin is a primary mechanism for regulating its activity on macrophages.
- This fragmentation results in plasmin derivatives with truncated or altered physiological functions.
- Understanding this regulation offers insights into macrophage-driven processes like inflammation and tissue repair.