Plasminogen is a complement inhibitor

Abstract

Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the complement cascade. Plasminogen binds the central complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both complement and coagulation, the two central cascade systems of a vertebrate organism. This complement-inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.

FIGURE 1.

Plasminogen binds to central human complement proteins C3 and C5. A, plasminogen binding to complement proteins C3 and C5 was analyzed by ELISA. C3 or C5 was bound to immobilized plasminogen, and bound proteins were detected using goat C3 or C5 antiserum and HRP-conjugated goat antiserum. B, binding of plasminogen to immobilized C3 and to the C3 activation fragments C3b, C3c, C3d, and C3a at equal molar concentrations was analyzed. Plasminogen bound to immobilized C3, C3b, C3c, and C3d but not to C3a. C, plasminogen used at concentrations ranging from 0.25 to 1.0 μg was bound to immobilized C3, C3b, and C3d at equal molar concentrations, and bound plasminogen was detected with goat plasminogen antiserum and HRP-conjugated goat antiserum; binding was dose-dependent. The bars represent the means of three independent experiments ±S.D. ***, p ≤ 0.001. D, plasminogen binding to C3 and C3 fragments was analyzed by Western blotting. Purified C3 and the C3 fragments C3b and C3c were separated by SDS-PAGE and transferred to a membrane, and the membrane was incubated with plasminogen. Bound plasminogen was detected with goat plasminogen antiserum. Plasminogen binding to C3b (E) and C3d (F) was analyzed by surface plasmon resonance. C3b or C3d was immobilized on the chip surface, and plasminogen was used as the analyte at concentrations ranging from 50 to 400 nm in the fluid phase. The results show a representative result of three independent experiments. w/o, without.

FIGURE 2.

Plasminogen-C3 interaction is mediated by lysine residues and affected by ionic strength, and plasminogen did not affect Factor H binding to C3b. A, the effect of the lysine analog ϵACA on plasminogen binding to C3, C3b, or C3d was analyzed by ELISA. Plasminogen bound to C3, C3b, and C3d in the absence of ϵACA was set as 100%. ϵACA reduced binding in a dose-dependent manner. The arrow indicates the concentration of 1 mm ϵACA. B, the effect of NaCl on plasminogen binding to immobilized C3, C3b, or C3d was assayed. Binding of plasminogen to C3, C3b, or C3d was set as 100%. NaCl reduced binding in a dose-dependent manner. The arrow indicates the physiological level (150 mm) of NaCl. Data show mean values of three experiments, and the S.D. is indicated by error bars. ***, p ≤ 0.001. The effect of ϵACA on plasminogen binding to immobilized C3b (C) or C3d (D) was analyzed by surface plasmon resonance. C3b or C3d was immobilized on the chip surface, and plasminogen with ϵACA was applied in the fluid phase. ϵACA reduced binding in a dose-dependent manner. Results of a representative experiment of three independent experiments are shown in C and D. E, plasminogen and Factor H bind to independent sites in the C3b protein. Factor H was combined with different amounts of plasminogen, the mixture was bound to immobilized C3b, and both bound Factor H and plasminogen were detected with specific antisera (i.e. goat Factor H and goat plasminogen). An antibody control is shown without a ligand (w/o). Data show mean values of four independent experiments, and S.D. is indicated by error bars. ***, p ≤ 0.001.

FIGURE 3.

Plasminogen binds to complement protein C5. A, plasminogen binding to C5 was assayed by ELISA. Plasminogen bound to immobilized C5 (black column 1), and in a reverse setting, C5 bound to immobilized plasminogen (black-white striped column 4). Bound proteins were detected with specific antiserum and HRP-conjugated goat antiserum. B, plasminogen used at increasing concentrations (0.25–1.5 μg) was bound to immobilized C5. Plasminogen bound to C5 in a dose-dependent manner. C, plasminogen binding to C5 was analyzed by surface plasmon resonance. C5 was immobilized on the chip surface, and plasminogen was used as the analyte at concentrations ranging from 50 to 400 nm in the fluid phase. The results show a representative result of three independent experiments. D, the effect of NaCl (■) and ϵACA (♦) on plasminogen binding to C5 was assayed by ELISA. Bound plasminogen was detected with goat plasminogen antiserum and HRP-conjugated goat antiserum. Plasminogen bound to C5 in the absence of either ϵACA or NaCl was set as 100%. NaCl and ϵACA reduced binding in a dose-dependent manner. The arrow indicates the physiological level (150 mm) of NaCl. The bars represent the means of three independent experiments, and the S.D. is indicated. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. w/o, without.

FIGURE 4.

Plasminogen enhances cofactor-assisted cleavage of C3b by Factor I. A, plasminogen used at 1, 5, or 10 μg was added to Factor H, Factor I, and C3b. The reaction mixture was separated by SDS-PAGE and transferred to a membrane, and C3b and C3b degradation fragments were visualized by Western blotting using goat C3 antiserum and HRP-conjugated goat antiserum. Intact C3b was detected as α′- and β-chains (lane 1). Cleavage of C3 was recorded by the reduced intensity of the 110-kDa α′ band and by the appearance of cleavage products α′68, α′43, and α′41 (lane 2). Plasminogen enhanced the C3b cleavage by Factor I and Factor H dose-dependently (lanes 3–5). B, densitometric analyses revealed that in the presence of 10 μg of plasminogen the intensity of the α′43 band was increased. A representative experiment of three is shown.

FIGURE 5.

Fluid phase and surface-bound plasmin cleaves C3b. A, plasminogen was activated by uPa in the fluid phase, and C3b was added. The reaction mixture was separated by SDS-PAGE and transferred to a membrane, and degradation of C3b was analyzed by Western blotting using goat C3 antiserum and HRP-conjugated goat antiserum. C3b degradation by plasmin was visualized by the appearance of the α′68, α′40, α′30, and α′17 bands (lane 2). The mobilities of the α′- and β-chains of the intact C3b protein are shown in lane 1. In the presence of ϵACA, aprotinin, or α₂-antiplasmin (α2-AP), C3b cleavage was reduced (lanes 3–5). Cleavage products generated by the complement serine protease Factor I in the presence of a cofactor (Factor H) were identified as α′68, α′43, and α′41 (lane 6). B, surface-bound plasmin degrades C3b. Plasminogen was immobilized, and uPa and C3b were added. At the indicated time, the supernatant was separated by SDS-PAGE and transferred to a membrane, and degradation of C3b was analyzed by Western blotting. Intact C3b was separated in lane 1. Surface-bound and activated plasmin cleaved C3b. Again, the cleavage products α′68, α′40, α′30, and α′17 were generated by plasmin (lanes 2–5). Cleavage was time-dependent. Neither plasminogen nor uPa alone degraded C3b significantly (lanes 6 and 7). A representative experiment of three is shown.

FIGURE 6.

Fluid phase and surface-bound plasmin cleaves C5. A, plasminogen was activated by uPa in the fluid phase, and C5 was added. The reaction mixture was separated by SDS-PAGE and transferred to a membrane, and degradation of C5 was analyzed by Western blotting using goat C5 antiserum and HRP-conjugated goat antiserum. C5 degradation by plasmin was visualized by the appearance of the α′50, α′45, α′30, and α′25 bands (lane 2). The mobilities of the α- and β-chains of the intact C5 protein are visualized in lane 1. In the presence of ϵACA, aprotinin, or α₂-antiplasmin (α2-AP), C5 cleavage was reduced (lanes 3–5). B, surface-bound plasmin degrades C5. Plasminogen was immobilized, and uPa and C5 were added. At the indicated time, the supernatant was separated by SDS-PAGE and transferred to a membrane, and degradation of C5 was analyzed by Western blotting. The mobility of the α- and β-chains of intact C5 are visualized in lane 1. Surface-bound and activated plasmin cleaves C5. The plasmin-mediated cleavage products α′65, α′50, α′45, α′30, and α′25 were detected (lanes 2–5). Cleavage was time-dependent. Neither plasminogen nor uPa alone significantly degraded C5 (lanes 6 and 7). A representative experiment of three is shown.

FIGURE 7.

Plasmin inhibits complement activation. Plasminogen activated by uPa was added to NHS, and each of the three complement pathways was activated by LPS, IgG, or mannan. Following incubation, terminal complement complex (TCC) formation was measured by ELISA using a specific terminal complement complex reacting antiserum. Plasmin inhibited AP (A), CP (B), and LP (C) activation in a dose-dependent manner. The arrows indicate the physiological level of plasmin(ogen). Used alone, neither plasminogen nor uPa showed strong effects. NHS was used as a positive control for activation according to the WiELISA kit. Buffer was used as a negative control. A representative experiment of three is shown, and the S.D. is indicated. ***, p ≤ 0.001.

FIGURE 8.

Plasmin inhibits complement-mediated hemolysis. A, the effect of plasmin on complement activation was analyzed using a hemolytic assay. Plasminogen was activated by uPa and added to NHS and rabbit erythrocytes. Erythrocyte lysis was evaluated by recording the release of hemoglobin at 414 nm. Total erythrocyte lysis was induced with H₂O and was set as 100%. uPa-treated plasminogen (♦) inhibited erythrocytes lysis, and the effect was dose-dependent. The arrow indicates the physiological concentration of plasmin(ogen). Neither plasminogen (■) nor uPa (▴) when added alone showed a significant inhibitory effect. In the presence of aprotinin (●), plasmin-mediated inhibition was blocked. B, plasmin inhibits both AP- and CP-mediated hemolysis. Plasminogen used at concentrations from 0.5 to 4 μm was activated by uPa, combined with NHS, and then added to antibody-sensitized sheep erythrocytes. Erythrocyte lysis was analyzed by measuring the optical density, and lysis induced by H₂O was set as 100%. The arrow indicates the physiological concentration of plasmin(ogen). C, α₂-antiplasmin (α2-AP) inhibited plasmin activity on sensitized sheep erythrocytes. The effect of α₂-antiplasmin was dose-dependent in the range of 25–100 μg/ml (columns 4–7). The bars represent the mean values of three independent experiments ±S.D. **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 9.

Plasmin inhibited C3a generation. A, the blocking effect of plasmin on complement activation in NHS was assayed by following C3a generation. Complement was activated via the AP by adding zymosan to NHS. Then plasmin(ogen) (5–10 μg), plasminogen (10 μg), uPa (0.2 μg), or Factor H (5 μg) was added. Samples were separated by SDS-PAGE and transferred to a membrane, and C3a generation was analyzed. No C3a was generated in NHS in the absence of zymosan (lane 1). Zymosan activated the AP, and C3a was generated (lane 2). uPa-treated plasminogen inhibited C3a generation (lanes 3 and 4). Plasminogen used alone did not inhibit C3a generation, uPa added alone had a minor blocking effect on C3a generation (lanes 5 and 6), and Factor H inhibited C3a generation (lane 7). C3a was used as a control (lane 8). B, C3a generation was also followed by ELISA. NHS in the absence of zymosan lacked C3a (column 1). Activation of NHS with zymosan resulted in the generation of C3a (black column 2). Again, plasmin used at 5 μg inhibited C3a generation (white striped column 3). Factor H also used at 5 μg inhibited C3a generation (gray striped column 4). Buffer represents the antibody control (column 5), and C3a was used as a positive control (column 6). The results are representative data of four independent experiments. **, p ≤ 0.01.

FIGURE 10.

Coagulation protease plasminogen controls multiple levels of complement cascade. Plasmin(ogen) affects the complement cascade at multiple levels. Plasminogen assists Factor H- and Factor I-mediated inactivation of C3b. In the presence of plasminogen, C3b inactivation is enhanced. In addition, when plasminogen is converted to the active protease plasmin, the protease can process the central complement components C3 and C5. The protease plasmin cleaves both C3 and C5a. The first cleavage generates C3b and biologically active C3a fragment or C5b and C5a (25). However, plasmin cleaves both C3b and C5b at additional sites and generates degradation fragments of C3b (C3b_deg) or C5b (C5_deg). In consequence, plasmin(ogen) blocks complement activation on multiple levels. Plasminogen, the proteolytically inactive zymogen, enhances C3b processing by the complement protease Factor I in the presence of the cofactor Factor H. Plasminogen binds to the central complement components C3b and C5 and when activated to plasmin cleaves and degrades C3b and C5. Plasminogen when activated to plasmin blocks all three major complement pathways; thereby, both plasminogen and in particular the active protease plasmin influence the activity of the complement cascade in terms of activation and inhibition. TCC, terminal complement complex.