Thromb Haemost 2015; 114(02): 277-288
DOI: 10.1160/TH14-06-0499
Coagulation and Fibrinolysis
Schattauer GmbH

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions

Mareike Kahl
1   Institute for Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany
,
Anke Gökçen
2   Fraunhofer Institute for Molecular Biology and Applied Ecology, Project Group “Bioresources”, Giessen, Germany
,
Silvia Fischer
1   Institute for Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany
,
Markus Bäumer
4   Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany
,
Jochen Wiesner
2   Fraunhofer Institute for Molecular Biology and Applied Ecology, Project Group “Bioresources”, Giessen, Germany
,
Günter Lochnit
1   Institute for Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany
,
Malgorzata Wygrecka
1   Institute for Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany
,
Andreas Vilcinskas
2   Fraunhofer Institute for Molecular Biology and Applied Ecology, Project Group “Bioresources”, Giessen, Germany
3   Institute for Phytopathology and Applied Zoology, Justus-Liebig-University, Giessen, Germany
,
Klaus T. Preissner
1   Institute for Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany
› Author Affiliations
Further Information

Publication History

Received: 06 June 2015

Accepted after major revision: 15 March 2015

Publication Date:
01 December 2017 (online)

Summary

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

 
  • References

  • 1 Beasley WD, Hirst G. Making a meal of MRSA-the role of biosurgery in hospital-acquired infection. J Hosp Infect 2004; 56: 6-9.
  • 2 Whitaker IS, Twine C, Whitaker MJ. et al. Larval therapy from antiquity to the present day: mechanisms of action, clinical applications and future potential. Postgrad Med J 2007; 83: 409-413.
  • 3 Grassberger M. Ein historischer Rückblick auf den therapeutischen Einsatz von Fliegenlarven. NTM Zeitschrift für Geschichte der Wissenschaften, Technik und Medizin 2002; 10: 13-24.
  • 4 Church JC. The traditional use of maggots in wound healing, and the development of larva therapy (biosurgery) in modern medicine. J Altern Complement Med 1996; 02: 525-527.
  • 5 Sherman RA. Maggot therapy for foot and leg wounds. Int J Low Extrem Wounds 2002; 01: 135-142.
  • 6 Fleischmann W, Grassberger M, Sherman RA. Maggot Therapy. Stuttgart, Thieme Verlag; 2003
  • 7 Sherman RA. Maggot therapy takes us back to the future of wound care: new and improved maggot therapy for the 21st century. DST 2009; 03: 336-344.
  • 8 Fleischmann W, Russ M, Moch D. et al. Biochirurgie – Sind Fliegenmaden wirklich die besseren Chirurgen?. Der Chirurg 1999; 70: 1340-1346.
  • 9 Sherman RA. Maggot versus conservative debridement therapy for the treatment of pressure ulcers. Wound Repair Regen 2002; 10: 208-214.
  • 10 Chambers L, Woodrow S, Brown AP. et al. Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds. Br J Dermatol 2003; 148: 14-23.
  • 11 Pavillard ER, Wright EA. An Antibiotic from maggots. Nature 1957; 180: 916-917.
  • 12 Mumcuoglu KY, Miller J, Mumcuoglu M. et al. Destruction of bacteria in the digestive tract of the maggot of Lucilia sericata (Diptera: Calliphoridae). J Med Entomol 2001; 38: 161-166.
  • 13 Bexfield A, Nigam Y, Thomas S. et al. Detection and partial characterisation of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus (MRSA). Microbes Infect 2004; 06: 1297-1304.
  • 14 van der Plas MJA, Dambrot C, Dogterom-Ballering HCM. et al. Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom. J Antimicrob Chemoth 2010; 65: 917-923.
  • 15 Kawabata T, Mitsui H, Yokota K. et al. Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment. Med Vet Entomol 2010; 24: 375-381.
  • 16 Horobin AJ, Shakesheff KM, Woodrow S. et al. Maggots and wound healing: an investigation of the effects of secretions from Lucilia sericata larvae upon interactions between human dermal fibroblasts and extracellular matrix components. Br J Dermatol 2003; 148: 923-933.
  • 17 Horobin AJ, Shakesheff KM, Pritchard DI. Promotion of human dermal fibrob-last migration, matrix remodelling and modification of fibroblast morphology within a novel 3D model by Lucilia sericata larval secretions. J Invest Dermatol 2006; 126: 1410-1418.
  • 18 Prete PE. Growth effects of Phaenicia sericata larval extracts on fibroblasts: Mechanism for wound healing by maggot therapy. Life Sci 1997; 60: 505-510.
  • 19 Zhang Z, Wang S, Diao Y. et al. Fatty acid extracts from Lucilia sericata larvae promote murine cutaneous wound healing by angiogenic activity. Lipids Health Dis 2010; 09: 24.
  • 20 Bexfield A, Bond AE, Morgan C. et al. Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis. Br J Dermatol 2010; 162: 554-562.
  • 21 van der Plas MJA, van der Does AM, Baldry M. et al. Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses. Microbes Infect 2007; 09: 507-514.
  • 22 van der Plas MJA, van Dissel JT, Nibbering PH. Maggot secretions skew monocyte-macrophage differentiation away from a pro-Inflammatory to a pro-angiogenic Type. PLoS ONE 2009; 04: e8071.
  • 23 Preissner KT, Wassmuth R, Muller-Berghaus G. Physicochemical characterization of human S-protein and its function in the blood coagulation system. Biochem J 1985; 231: 349-355.
  • 24 Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096.
  • 25 Osterud B, Flengsrud R. Purification and some characteristics of the coagulation factor IX from human plasma. Biochem J 1975; 145: 469-474.
  • 26 Emsley J, McEwan PA, Gailani D. Structure and function of factor XI. Blood 2010; 115: 2569-2577.
  • 27 Schmidtchen A, Wolff H, Rydengard V. et al. Detection of serine proteases secreted by Lucilia sericata in vitro and during treatment of a chronic leg ulcer. Acta Derm Venereol 2003; 83: 310-311.
  • 28 Fujikawa K, Chung DW, Hendrickson LE. et al. Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein. Biochemistry 1986; 25: 2417-2424.
  • 29 Ratnoff OD, Pensky J, Ogston D. et al. The inhibition of plasmin, plasma kallik-rein, plasma permeability factor, and the C’1r subcomponent of the first component of complement by serum C’1 esterase inhibitor. J Exp Med 1969; 129: 315-331.
  • 30 Pensky J, Levy LR, Lepow IH. Partial purification of a serum inhibitor of C’1-esterase. J Biol Chem 1961; 236: 1674-1679.
  • 31 Van Nostrand WE, McKay LD, Baker JB. et al. Functional and structural similarities between protease nexin I and C1 inhibitor. J Biol Chem 1988; 263: 3979-3983.
  • 32 Meijers JCM, Vlooswijk RAA, Bouma BN. Inhibition of human blood coagulation factor XIa by C.hivin.1 inhibitor. Biochemistry 1988; 27: 959-963.
  • 33 Hagedorn I, Schmidbauer S, Pleines I. et al. Factor XIIa inhibitor recombinant human albumin Infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding. Circulation 2010; 121: 1510-1517.
  • 34 Bjorkqvist J, Jamsa A, Renne T. Plasma kallikrein: the bradykinin-producing enzyme. Thromb Haemost 2013; 110: 399-407.
  • 35 Colman RW. Activation of plasminogen by human plasma kallikrein. Biochem Biophys Res Commun 1969; 35: 273-279.
  • 36 Mandle Jr. R, Kaplan AP. Hageman factor substrates. Human plasma prekallik-rein: mechanism of activation by Hageman factor and participation in hageman factor-dependent fibrinolysis. J Biol Chem 1977; 252: 6097-6104.
  • 37 Goldsmith Jr. GH, Saito H, Ratnoff OS. The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments. J Clin Invest 1978; 62: 54-60.
  • 38 Mandle Jr. RJ, Kaplan AP. Hageman-factor-dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen. Blood 1979; 54: 850-862.
  • 39 van der Plas MJA, Andersen AS, Nazir S. et al. A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis. PLoS ONE 2014; 09: e92096.
  • 40 Cazander G, Schreurs MW, Renwarin L. et al. Maggot excretions affect the human complement system. Wound Repair Regen 2012; 20: 879-886.
  • 41 Broughton 2nd G, Janis JE, Attinger CE. The basic science of wound healing. Plast Reconstr Surg 2006; 117 Suppl 12S-34S.
  • 42 Chavakis T, Kanse SM, Pixley RA. et al. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J 2001; 15: 2365-2376.
  • 43 Frick IM, Akesson P, Herwald H. et al. The contact system: A novel branch of innate immunity generating antibacterial peptides. EMBO J 2006; 25: 5569-5578.
  • 44 Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 2013; 13: 34-45.
  • 45 Čeřovský V, Žárek J, FuČík V. et al. Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata . Cell Mol Life Sci 2010; 67: 455-466.
  • 46 Heuer HFleck. Seraticin-ein antibiotischer Wirkstoff von Fliegenlarven. Deutsche Apotheker Zeitung 2010; 150: 394-395.
  • 47 Cazander G, Pritchard DI, Nigam Y. et al. Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: Larval secretions contain molecules that accelerate wound healing, reduce chronic inflammation and inhibit bacterial infection. Bioessays 2013; 35: 1083-1092.
  • 48 Sherman RA. Mechanisms of maggot-induced wound healing: What do we know, and where do we go from here?. Evid Based Complement Alternat Med 2014; 2014: 592419.