Figure 1.
Thrombin-derived peptides modulate the cytokine response to LPS in vitro.
(A and B) Measurement of nitrite release of RAW 264.7 macrophages stimulated with 10 ng/ml E. coli or P. aeruginosa LPS in combination with the indicated concentrations of GKY25 and HVF18 (n = 3). (C and D) RAW 264.7 cells were stimulated with 10 ng/ml E. coli LPS and cytokines were analysed in the cell supernatants (n = 3). (E and F) Human blood was treated with 10 ng/ml E. coli LPS alone, or with LPS and GKY25 (E) or HVF18 (F). After 6 h of incubation TNF-α was determined in plasma by ELISA (n = 3). (G) Binding of GKY25 and WFF25 to 125I-labeled E. coli LPS using a slot blot assay. (H) Stimulation of RAW 264.7 cells by LPS and the effects of peptides. Left panel: LPS was incubated with GKY25 or WFF25, followed by addition of serum and incubation with the cells. Right panel: Standard procedure as in A. Peptides were added to cells pre-incubated briefly with LPS in DMEM with 10% FBS (n = 3).
Figure 2.
Thrombin-derived peptides influence the coagulation system.
(A+B) The activated partial thrombin time (aPTT), prothrombin time (PT) and thrombin clotting time were determined after addition of buffer (control) or 20 µM of GKY25, HVF18 or WFF25 to human citrate plasma (CP) or 50 µM peptide in mouse CP (n = 3). (C) Whole blood was incubated with 10 ng/ml E. coli LPS with or without the indicated peptides at different concentrations. The clotting reaction in recalcified human CP was initiated by adding the blood cells. (Significance relative the LPS-treated sample is presented. n = 3) (D) Human monocytes were stimulated with 100 ng/ml E. coli LPS with or without the indicated peptides. The clotting reaction was initiated by adding LPS- and/or peptide-treated monocytes to human CP supplemented with CaCl2. (Significance relative the LPS-treated sample is presented. n = 3) (E) FACS analysis of TF expression on human CD14-positive monocytes treated with peptides and/or LPS (control; sodium citrate buffer, GKY25 only; indicates 10 µM peptide, HVF18 only; 40 µM peptide; n = 3).
Figure 3.
Anti-inflammatory effects of thrombin derived peptides in vivo.
(A–D) Septic shock in C57BL/6 mice was induced by i.p. injection of 18 mg/kg E. coli LPS. Thirty minutes after LPS injection, GKY25, WFF25, HVF18 (0.5 mg/mouse) or buffer were administered i.p. (A) Survival was followed for 7 days (n = 10–19/group (p<0.0001)). (B) Weight of the surviving animals. (C) Cytokines measured in blood from animals sacrificed at 8 h or 20 h after LPS injection (8 h: n = 8–12/group; 20 h: n = 10–14/group). (D) Light microscopy pictures (20× magnification, scale bar: 100 µm) of representative mouse lung tissue from control mice (buffer only), LPS and buffer or LPS and peptide treated mice 20 h after LPS injection. (E) C57BL/6 mice were injected i.p. with 36 mg/kg P. aeruginosa LPS followed 30 minutes later by i.p. administration of 0.5 mg of GKY25. After 20 h mice were sacrificed and cytokine levels in blood were determined (n = 8–10/group).
Figure 4.
Modulation of coagulation in vivo.
(A–D) C57BL/6 mice were injected i.p. with 18 mg/kg E. coli LPS to induce an endotoxin mediated shock followed by treatment with buffer, GKY25 or WFF25 (0.5 mg/mouse). (A) Animals were sacrificed after 20 h or 7 days and platelet numbers were determined (n = 12–20/group) (#: indicates no animals reached that time point, see Fig. 3A) (B) Quantification of thrombin/antithrombin complexes by ELISA (n = 13–20/group). (C) Comparison of lung lesions in sections 20 h after LPS injection. Scanning electron micrographs show representative mouse lung sections (scale bar: 50 µm). (D) Prothrombin time (PT) (n = 9–14/group) and activated partial thromboplastin time (aPTT) (n = 5–11/group) were measured in citrate plasma isolated from mice 20 h after injection of indicated substances. (***p<0.001).
Figure 5.
GKY25 modulates NO release in response to bacterial products.
RAW 264.7 macrophages were stimulated with E. coli (A) or P. aeruginosa (B) culture supernatants (Sup) or heat killed (HK) bacteria in the presence of GKY25 and NO release was determined using the Griess reaction.
Figure 6.
Effects of GKY25 during P. aeruginosa sepsis.
Mice were infected with P. aeruginosa (5×109 cfu/ml) i.p. followed by administration of buffer or GKY25 (0.5 mg) sc. after 1 h (1×), or 1 and 7 h (2×) after infection. (A–C) Mice were sacrificed 12 h after infection. (A) Analysis of bacterial counts in the indicated organs (n = 5–10/group). (B) Cytokine analyses in plasma (n = 8–12/group). (C) Scanning electron micrographs of representative mouse lung tissues. Non-infected mice were used as negative control (scale bar: 20 µm). (D). Survival was monitored for 7 days (n = 9–13/group; **p<0.01).