Molecular intercommunication between the complement and coagulation systems

Abstract

The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

FIGURE 1

Thrombin-induced cleavage of C3 and C5. Human C3 (5 μg; concentration 100 μg/ml) was incubated in DPBS in the absence or presence of increasing concentrations of human thrombin for 90 min. A, Western blot analysis of the C3 cleavage product in the presence of thrombin using polyclonal anti-C3a IgG as detection Ab. Equal protein loading was ensured for all samples. Depicted blot is representative of n = 3 experiments. B, ELISA assessment for C3a from samples coincubated with thrombin and C3 in DPBS (n = 3). C, C3 (100 μg/ml) and thrombin (5 μg/ml) were incubated as a function of time with subsequent evaluation for C3a by ELISA. D, Western blot analysis of C5 cleavage in the presence of thrombin. *p < 0.05 versus control.

FIGURE 2

FXa-mediated C3 and C5 cleavage with subsequent generation of C3a and C5a. Human C3 or C5 (5 μg; concentration 100 μg/ml) were incubated in DPBS in the absence or presence of increasing concentrations of FXa for 90 min at 37°C. A, Western blot detection of C3a following exposure of C3 to FXa. Equal protein loading was ensured for all samples. Blots are representative of at least three independent experiments. B, ELISA evaluation for C3a following coincubation of FXa and native C3. *p < 0.05 versus control (n = 3). C, Chemotaxis assay using HMC-1 cells to determine biological activity of C3-cleavage product postexposure to FXa (n = 6). Recombinant human C3a (100 ng/ml) served as a positive control. D, Western blot analysis of samples following exposure of C5 to FXa using anti-C5a IgG. Nonglycosylated, rC5a served as a positive control. Blot is representative of three independent experiments. E, ELISA assessment of the preparations described in D. *p < 0.05 versus control. F, Evaluation of C5 + FXa preparations for chemotactic activity using human neutrophils. Recombinant human C5a (100 ng/ml) served as a positive control. For each experimental condition, n = 6.

FIGURE 3

MS analysis of FXa-induced cleavage products of C3 and C5. MALDI-TOF MS analysis of the C3 and C5 cleavage products following FXa incubation. A, Mass sizes of C3a produced by FXa-induced C3 cleavage. B, Mass sizes of deglycosylated, recombinant human C5a (upper panel) and C5a generated by incubation of C5 with FXa (lower panel).

FIGURE 4

Plasmin-induced C3 and C5 cleavage. Human C3 or C5 (each at 5 μg; concentration 100 μg/ml) were incubated in DPBS in the absence or presence of increasing concentrations of plasmin for 90 min (37°C). A and D, Western blot analysis of C3 or C5 samples following exposure to plasmin. As a positive control, 50 ng of rC3a or rC5a was used. Depicted blots are representative of n = 3 independent experiments. B and E, C3a- or C5a-ELISA measurements of C3 or C5 samples after plasmin incubation (n = 3). C, Analysis of chemotactic activity of C3 preparation following incubation with increasing amounts of plasmin using HMC-1 cells. Recombinant human C3a (100 ng/ml) served as positive control (n = 6). F, Following incubation of C5 with increasing amounts of plasmin, samples served as chemotactic stimulus for isolated human neutrophils. C5a (100 ng/ml) was used as a positive control. n = 6 per experimental condition. *p < 0.05 versus control.

FIGURE 5

C3-and C5-proteolytic activity of various coagulation factors (FXa, FIXa, FXIa, plasmin, thrombin). A and B, C3- and C5-proteolytic activity of various coagulation/fibrinolysis factors (each at 80 nM) were assessed by ELISA measurements of C3a and C5a generated within 90 min in the presence of increasing concentrations of the substrate (10, 54, 108, 270, 540, 810, and 1080 nM C3 or C5). Every proteolytic activity value represents the average of duplicate measurements based on five independent experiments.

FIGURE 6

FXa-induced serum complement activation. Human serum was incubated in the presence or absence of increasing concentrations of FXa for 90 min at 37°C. Evaluation of serum samples for C3a (A) and C5a (B) concentration following FXa-incubation using ELISA analysis. *p < 0.05 versus control (n = 3). C, CH50 was assessed in human serum in the presence or absence of 100 μg/ml FXa. Data are representative of 12 separate and independent experiments. *p < 0.05 versus control. ELISA analysis of serum samples for C5a postincubation with FXa (40 μg/ml) in the copresence of increasing amounts of the FXa-selective inhibitors sodium fondaparinux (D) or sodium enoxaprin (E). *p < 0.05 versus control; n = 6. F, Correlation analysis between generated TAT complexes and complement activation product C5a in the plasma of patients early (<1 h) after multiple injury.

FIGURE 7

Simplified model of the serine protease system. Depiction of the complex interplay between the coagulation/fibrinolysis cascades and the complement system. The serine proteases of the complement, coagulation, and fibrinolysis systems are all highlighted in yellow. The black dotted arrow bars show previously known interactions of these systems. The red arrows identify the new paths of complement activation by the coagulation/fibrinolysis factors resulting in the generation of C3a and C5a. aPC, activated protein C; MAC, membrane attack complex; MBL, mannan-binding lectin; PK, prekallikrein.