Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism

Abstract

Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation.

Keywords: aggregation; host defense peptides; lipopolysaccharides; thrombin.

Fig. S1.

Characteristics of rTCP₉₆. (A) Western blot analysis illustrating α-thrombin (α-T; 10 μM), α-thrombin digested for 3 h by HNE (α-T + HNE; 0.8 μg/mL), GKY25 (10 μM), and rTCP₉₆ (10 μM). The arrow indicates 11-kDa TCPs. This fragment has been previously characterized and is shown here for comparison and clarity (10). (B) Production and purity of rTCP₉₆ was analyzed via SDS/PAGE and Western blotting. (C) Comparison of heparin binding of rTCP₉₆ and γ-thrombin (γ-T) using heparin-affinity fast protein liquid chromatography. (D–F) Gel-based overlay assay and RDA revealed inhibition zones using 10 μM of both GKY25 and rTCP₉₆ in Escherichia coli (Ec) and GKY25 in Ec and Pseudomonas aeruginosa (Pa) (n = 3). (G) VCA demonstrated the antimicrobial activity of 3 μM rTCP₉₆ and 3 μM GKY25 (a positive control) (n = 4). (H) In a separate experiment, a concentration-dependent antibacterial effect of rTCP₉₆ was observed at or above 0.5 μM (n = 4). (I) rTCP₉₆ (up to 10 μM) did not demonstrate any hemolytic effects. (J) Kinetic analyses of rTCP₉₆, GKY25, and IVE25 binding to surface-localized LPS are presented. (K) Slot-blot assay demonstrated binding of rTCP₉₆ (2 μg and 5 μg) to biotin-labeled LPS. The binding was blocked after the addition of heparin. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 1.

Analysis of TCP–LPS interactions and LPS-mediated aggregation. (A) Ellipsometry analysis revealed adsorption of 1 μM rTCP₉₆, GKY25, and IVE25 at LPS-coated surfaces from 10 mM Tris (pH 7.4) (transformation to milligrams of peptide bound per milligram of LPS was achieved by dividing by the amount of LPS preadsorbed: 1.48 ± 0.38 mg/m²). (B) We used an MST assay to quantify the interaction of rTCP₉₆ with LPS. The peptide IVE25 was used as a negative control. The mean values of four measurements ± their SDs are shown. (C) Changes in the secondary structure of rTCP₉₆ triggered by LPS binding were analyzed by CD spectroscopy. The data showed an increase in the β-sheet structure in rTCP₉₆ (10 μM) and an increase in the α-helical content in GKY25 (10 μM) after 30 min of incubation with LPS (100 μg/mL) at 37 °C. (D) ThT assay, identifying a significant increase in amyloid formation in rTCP₉₆ (at 10 μM) after the addition of 100 μg/mL LPS (n = 3). au, arbitrary units. ***P < 0.001, ****P < 0.0001. (E) Negative-stain TEM revealed the formation of aggregates after incubation of rTCP₉₆ with LPS in 10 mM Tris (pH 7.4) and supplemented with citrated plasma (CP). (Insets) Same samples without LPS treatment using the same magnification. Structural changes in digested α-thrombin (α-T; 10 μM) by HNE and γ-thrombin (γ-T) were recorded via ThT assay (F) and CD spectroscopy (G). Thrombin was used as a control. (H and I) TEM analysis of aggregates (1–2 μm in length) present in digested α-T and γ-T (0.1–0.5 μm in length) after incubation with LPS. Only small amounts of aggregates, or none at all, were detected in α-T alone, or after addition of LPS, respectively.

Fig. S2.

Analyses of aggregation and colocalization of TCPs. (A) Effects of GKY25 at the indicated doses on LPS-induced rTCP₉₆ aggregation (100 μg/mL LPS). The dotted line indicates the fluorescence of pure rTCP₉₆ (10 μM; n = 4). (B) Dose dependence of rTCP₉₆ aggregation, which was triggered by 100 μg/mL LPS, is demonstrated in the presence of 10 mM Tris (pH 7.4) (Left, n = 4) or 10 mM Tris (pH 7.4) supplemented with 1% citrated human plasma (Right, n = 4). (C) DLS measurements were performed to determine the hydrodynamic radii of 1 μM rTCP₉₆, 10 μg/mL LPS, and LPS-treated rTCP₉₆ (n = 5). GKY25 was used as a negative control. (D) Far-UV CD spectra of rTCP₉₆ (10 μM) in the presence and absence of LPS (100 μg/mL) in Tris buffer were recorded after incubation for 10 min and 120 min. ThT assay (E) and DLS analyses (F) were performed of rTCP₉₆ (10 μM) with or without LPS (100 μg/mL) addition after the indicated incubation times (n = 4). (G) Western blot analysis of rTCP₉₆ [at the indicated concentrations (2 μL) and in wound fluids from patients 1 and 2 (2 μL)]. (Upper) Representative image showing the molecular weight region used for quantification (Materials and Methods) of 11-kDa TCPs is presented. (Lower) Obtained concentrations relative the standard curve (n = 4). (H) Analysis of clustering and colocalization patterns of TCPs and LPS. The histogram depicts PCCF function values for intervals of distances. One microscopic image from each sample was analyzed. A PCCF value above 1 corresponds to nonrandom mutual distribution-colocalization of two types of particles (24). au, arbitrary units; R_h, hydrodynamic radius. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

In silico studies. (A) Structure of the 96-aa TCP after 100 ns of atomic resolution molecular dynamics simulations. The hydrophobic residues are shown in gray. The formation of a new hydrophobic cluster (yellow, labeled residues) of the free TCP between the helix segment 80–96 (red) and the sheet residues 46–60 (brown) relies on a twist within the sheet. The extended tail residues formed by residues 46–60 and 80–96 preferentially interact with the lipid tails. The flat patch composed of residues 1–20 (blue) of the 96-aa TCP preferentially interacts with the polar head of LPS. (B) Snapshot from a TCP aggregation simulation showing a representative aggregated state that is mediated by LPS molecules (purple, thick lines). The TCP backbones are shown in various colors (thin lines). It is apparent that LPS intercalates within the TCP clusters and predominantly connects the preferential interaction sites. (C) Aggregation studies of TCP in the presence (+LPS) and absence (−LPS) of LPS. The panels indicate intermolecular distances of all eight TCP molecules in the simulation box. Bright areas indicate contact with larger bright segments signifying larger contact areas between molecules. Dark areas indicate that the respective molecules do not interact. Increased aggregation is apparent through the appearance of additional bright areas in the system containing LPS.

Fig. S3.

Computational prediction of coaggregation of TCP and LPS. Interactions of eight 96-aa TCP molecules and LPS from five random starting orientations were predicted.

Fig. 3.

Coaggregation of E. coli and TCPs. (A) Confocal microscopy and ThT staining were used to detect E. coli-induced amyloid formation of rTCP₉₆ (10 μM) in 10 mM Tris buffer (pH 7.4). (B) TEM with negative stain analysis reveals the aggregation of rTCP₉₆-treated bacteria. (C) Fluorescence microscopy shows the agglutination of permeabilized rTCP₉₆-treated bacteria after the addition of FITC dye. (Inset) GKY25-treated E. coli (Ec). (D) Significant increase in the number of bacteria per aggregate was detected after treatment with 10 μM rTCP₉₆ compared with GKY25 (n = 3). **P < 0.01. (E) Phagocytosis assay using the macrophage cell line RAW 264.7 revealed a significant increase in the phagocytosis of E. coli particles pretreated with rTCP₉₆ (0.5 μM; n = 4). **P < 0.01. (F) Confocal microscopy analysis of phagocytized E. coli particles (green) pretreated with rTCP₉₆ (Alexa 568, red); we used RAW 264.7 cells (DAPI, blue). DIC, differential interference contrast.

Fig. S4.

Control microscopy analysis. Control confocal microscopy analysis of phagocytized E. coli particles (green) and rTCP₉₆ (Alexa 568, red) incubated with RAW 264.7 cells (DAPI, blue).

Fig. 4.

C-terminal fragments of thrombin are found in human acute wound fluids, fibrin sloughs, and wound dressings. (A) Western blotting yielded 11-kDa TCPs in all wound fluid (WF) samples (arrow): (1) rTCP₉₆ (10 μM, 1 μL), (2) rTCP₉₆ in plasma (10 μM, 1 μL), (3–7) WF from patients 1–5 (patient 1, 80 μg total protein per lane; patients 2–5, 120 μg total protein per lane). (B) Ex vivo, aggregates were detected by TEM and demonstrated aggregated endogenous TCPs by using gold-labeled anti-VFR17 epitope IgG (blue dots) in WF after incubation with LPS (Left; 100 μg/mL) or without LPS (Right). (Insets) Display of a 50× lower magnified overview of the same samples. (C) Formation of aggregates in WF triggered by E. coli bacteria. (Inset) WF only, using the same magnification. Confocal microscopy, using primary antibodies against TCPs followed by Alexa 568-labeled secondary antibodies and ThT staining, was used to determine amyloid aggregates containing TCPs in the WF in the presence of LPS (D) or E. coli (E). (F) Western blotting of extracts from fibrin slough (FS) and wound dressing revealed the presence of 11-kDa TCP fragments in both samples (arrow): (1) rTCP₉₆, (2) rTCP₉₆ in CP, (3) extract of FS, and (4) extract of wound dressing. (G) Colocalization of endogenous TCPs and LPS in the FS from two patients was detected via TEM analysis. The TCPs were recognized by anti-rabbit IgG with labeled gold particles 10 nm in diameter (blue dots). We detected LPS using IgG against LPS with labeled gold particles 5 nm in diameter (red dots). Colocalization exceeding 90% was observed in both samples. (Insets) Same samples using a fourfold higher magnification.

Fig. 5.

Multiple effects of thrombin and its fragments. Injury and infection activate the coagulation cascade resulting in Factor X (FX)-mediated cleavage of prothrombin. Thrombin is further cleaved by HNE, generating TCPs, or autoproteolysed (Trb) (39). Further proteolysis by endogenous HNE or bacterial elastases such as lasB from P. aeruginosa (PAE) gives rise to short TCPs (references in parentheses). The red-colored frame indicates the findings presented in this work.