Wound Fluid from Venous Leg Ulcers Degrades Plasminogen and Reduces Plasmin Generation by Keratinocytes  Richard Hoffman, Sue Starkey, Jane Coad  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1140-1144 (December 1998) DOI: 10.1046/j.1523-1747.1998.00429.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Degradation of plasminogen by wound fluids. Wound fluids (2 μl) were incubated with Sigma plasminogen (0.4 mg per ml) in PBS (total volume 20 μl) for 45 min at 37°C. Samples were run on 12% SDS-PAGE, blotted, and probed with (a) an anti-plasminogen antibody and (b) an anti-K1–3 antibody. Plg, plasminogen; K1–3, kringle domains 1–3; WF, wound fluids. The positions of molecular weight markers are shown on the left. Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Time course degradation of plasminogen by leg ulcer fluid B. Wound fluid B (2 μl) was incubated with Sigma plasminogen (0.4 mg per ml) in PBS (total volume 20 μl) for 5, 10, 20, 30, 60 min, or 18 h at 37°C. Samples were run on 12% SDS-PAGE, blotted, and probed with an anti-plasminogen antibody. Plg, plasminogen; K1–3, kringle domains 1–3; WFB, undiluted wound fluid B alone. The positions of molecular weight markers are shown on the left. Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of protease inhibitors on degradation of plasminogen by leg ulcer fluid B. Wound fluid B (2 μl) was preincubated alone or in the presence of PMSF (1 mM) and/or 1,10 phenanthroline (5 mM) for 10 min at 37°C, and Sigma plasminogen (0.4 mg per ml) (total volume 20 μl) was then added for 60 min. K1–3, kringle domains 1–3; Plg, plasminogen; CON, wound fluid B incubated with plasminogen; Phe, wound fluid B incubated with plasminogen and 1,10 phenanthroline; PMSF, wound fluid B incubated with plasminogen and PMSF; Phe PMSF, wound fluid B incubated with plasminogen, PMSF, and 1,10 phenanthroline; SOLV, wound fluid B incubated with plasminogen, dimethylsulfoxide, and ethanol equivalent to their amounts in the inhibitors. Samples were run on 12% SDS-PAGE, blotted, and probed with an anti-plasminogen antibody. The positions of molecular weight markers are shown on the left. Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of α1 anti-trypsin on neutrophil elastase activity in wound fluid B. Wound fluid B (2 μl) was preincubated alone or with α1 anti-trypsin for 10 min at 37°C, and then incubated with the neutrophil elastase substrate N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (final concentration 0.2 mM) as described inMaterials and Methods. Change in optical density at 405 nm was measured at intervals. Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of α1 anti-trypsin and α2 anti-plasmin on degradation of plasminogen by leg ulcer fluid B. Wound fluid B (2 μl) was preincubated alone or with α1 anti-trypsin or α2 anti-plasmin for 10 min at 37°C, and then incubated with Calbiochem plasminogen (0.4 mg per ml; total volume 20 μl) for a further 60 min. K1–3, kringle domains 1–3; Plg, plasminogen; CON, wound fluid B incubated with plasminogen;α1AT, wound fluid B incubated with plasminogen and α1 anti-trypsin;α2AP, wound fluid B incubated with plasminogen and α2 anti-plasmin. Samples were run on 12% SDS-PAGE, blotted, and probed with an anti-plasminogen antibody. The positions of molecular weight markers are shown on the left. Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of preincubating plasminogen with leg ulcer fluid on subsequent generation of plasmin (a) in the presence of HaCaT cells (b) in a cell-free system. (a) HaCaT cells were plated in 96 well microtitre plates as described inMaterial and Methods. Plasmin substrate was then added together with the following additions and optical density at 405 nm was determined at intervals. ○, Plasminogen;•, plasminogen and wound fluid B;□, plasminogen preincubated with wound fluid B for 10 min;▪, plasminogen preincubated with wound fluid B for 60 min;▵, wound fluid B alone; s, no further additions. Data are from a representative experiment that was repeated twice with similar results. (b) Plasmin substrate was added to wells in 96 well microtitre plates and additions were made as described in (a). Journal of Investigative Dermatology 1998 111, 1140-1144DOI: (10.1046/j.1523-1747.1998.00429.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions