Figure 5: In vivo relevance of FYT21.

(a) Wound fluids from non-healing leg ulcers (numbers 1–5) were analysed by western blotting using an antibody recognizing the C-terminal part of thrombin; plasma (p) is shown in lane 2. (b) An EM picture of a leukocyte, located in fibrin slough from an infected chronic wound, containing C-terminal epitopes of thrombin (black dots). The scale bars indicate 2 μm (left) and 100 nm (right). (c) Representative LC–MS/MS chromatogram of affinity purified wound dressing extract depicted in black; HVF18 eluted at 54 min and FYT21 at 59 min. The Orbitrap mass spectra of the triple-charged HVF18 peptide (m/z 757.7625) and the quadruple-charged FYT21 peptide (m/z 671.3670) enabled mass determination of both peptides (isotopic distribution depicted in grey). The assigned fragmentation pattern of HVF18 is given in red. (d) Cytokine analysis of blood collected from Balb/c mice (female 10–12 weeks), 4 h after i.p. injection with 1 mg kg⁻¹ of LPS or PBS, which was followed after 30 min with injection of FYT21 (500 μg) or 10 mM Tris, pH 7.4. Control mice were injected twice with buffer. Results are means±s.e.m. of three independent experiments with 3–6 mice per group. (e) In vivo image of mice injected s.c. with the reactive oxygen species reporter probe L-012, 4 h after i.p. injection with 5 mg kg⁻¹ of LPS and 500 μg FYT21 as described in d. Results are means±s.e.m. of three independent experiments with 3 mice per group. (f) A representative image of the four groups at 2 h and 4 h after injection: C, control; FYT, FYT21; L+F, LPS+FYT21. Values are significantly (*P<0.05, **P<0.005 and ***P<0.0005) different from the controls as analysed using a Mann–Whitney test.