Regular ArticleLonofibrase, a novel α-fibrinogenase from Lonomia obliqua caterpillars
Introduction
Lepidopterous larvae are well known for causing accidents upon skin contact with their bristles. The resulting clinical profile varies according to the species involved as well as the victim's physical condition. In human beings, the accidents can vary from a simple burning sensation to a severe envenomation state with several lethal cases [1]. The Saturniidae family, especially the Lonomia genus, presents several examples of such dangerous species.
A severe and prolonged bleeding syndrome caused by Lonomia achelous caterpillars' envenomation was first reported by Arocha-Piñango and Layrisse [2] in Venezuela. Among other blood coagulation disturbances, intense fibrinolytic activity was found in the patient's plasma. Plasmin-like and urokinase activities were characterized with synthetic chromogenic substrates, and fibrinolytic activity upon human blood clot was also found in the crude venom and other caterpillars extracts [3], [4].
In Brazil, similar cases of envenomation have been described for Lonomia obliqua, which was first reported in the states of Santa Catarina and Rio Grande do Sul [5], [6], [7]. The clinical profile resulting from L. obliqua's envenomation includes central and peripheral hemorrhage, hematuria, disseminated intravascular coagulation, acute renal failure, and, frequently, death of untreated patients. Envenomed patients have been treated with a polyclonal horse antilonomic serum [8], [9]. As a consequence of the massive hemorrhagic disorder, envenomed individuals present decreased plasma fibrinogen, factor V, factor XIII, α2-antiplasmin, and plasminogen, and increased thrombin activity, factor VIII:c, von Willebrand factor, fibrinolytic activity, and fibrinogen degradation products [5], [9], [10], [11], [12]. Most of these alterations are consistent with the dramatic hemorrhagic pattern following envenomation. However, it has been reported that the bristles extract of L. obliqua caterpillar also contains a factor X activator [13] as well as a prothrombin activator, the latter being named LOPAP (L. obliqua prothrombin activator protease) [14]. Since no fibrinolytic activity has been detected in the bristles extract, it has been proposed that LOPAP induces a severe consumption coagulopathy, thus producing the observed hemorrhagic syndrome [14], [15]. Contrary to this, other reports indicate the presence of a potent fibrinogenolytic activity in the extracts obtained from both caterpillar species [16], [17], [18]. Since the accidents with these gregarious animals usually implicate contact with a whole colony with several individuals being smashed, the obligatory skin contact of the victim with other caterpillars' secretions raises the possibility that several active principles from different insect sources are involved in the envenomation syndrome.
In the present work, we describe the isolation and characterization of a potent fibrinogenolytic enzyme (lonofibrase) from the material obtained from the secretory system of the L. obliqua caterpillars. Lonofibrase rapidly degrades fibrinogen into unclottable derivatives, consistent with the hemorrhagic profile observed upon envenomation.
Section snippets
Reagents
Bovine fibrinogen, benzoyl-dl-arginine p-nitroanilide (dl-BAPNA), and plasmin were obtained from Sigma (St. Louis, MO, USA). Thrombin was purified from human plasma by the method described by Ngai and Chang [19]. Citrated plasma was obtained from Hospital de Clı́nicas (UFRGS) and Hospital São Lucas (PUCRS). All other chemicals were of the highest purity commercially available. Mono Q HR 5/5 and Superdex 75 HR 10/30 columns were from Amersham (Sweden).
Venom extraction
Caterpillars from different colonies were
Fibrinogenolytic activity
Fig. 1 shows that the crude L. obliqua secretion decreased the rate of thrombin-induced fibrinogen clotting in a dose-dependent manner. Similar results were obtained in experiments using whole plasma (data not shown). Furthermore, fibrinogen became unclottable when treated with 20 μg of the crude material. It is important to emphasize that none of the L. obliqua secretion concentrations tested caused direct fibrinogen clotting.
The effect of caterpillar secretion towards purified fibrinogen was
Discussion
The occurrence of enzymes affecting hemostasis has been described thoroughly in several organisms. Some of these enzymes have as target a common pathway of coagulation cascade, acting as procoagulant as well as anticoagulant active principles. Among these principles, there are several fibrin(ogen)olytic enzymes mainly from snake venoms, such as α- and β-fibrinogenases from Vipera lebetina [23], [24]; α- and β-fibrinogenases from Trimeresurus mucrosquamatus [25]; α-fibrinogenase from Agkistrodom
Acknowledgements
We would like to thank Dr. Robson Monteiro from Universidade Feredal do Rio de Janeiro (UFRJ). We also acknowledge Centro de Informações Toxicológias (CIT) and the Fire Department of Erechim in the state of Rio Grande do Sul. This work was supported by the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).
References (43)
- et al.
Fibrinolysis produced by contact with a caterpillar
Lancet
(1969) - et al.
Lonomia genus caterpillar toxins: biochemical aspects
Biochimie
(2000) - et al.
Intracerebral haemorrhage after contact with Lonomia caterpillars
Lancet
(1996) - et al.
A Ca++ activated serine protease (LOPAP) could be responsible for the haemorrhagic syndrome caused by the caterpillar Lonomia obliqua
Lancet
(1999) - et al.
A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (LOPAP) biochemical characterization
Thromb. Res.
(2001) - et al.
Biochemical characterization of Basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus
Arch. Biochem. Biophys.
(1995) - et al.
Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom
Toxicon
(2002) - et al.
β-Fibrinogenase from the venom of Vipera lebetina
Toxicon
(1991) - et al.
α-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom
Toxicon
(1983) - et al.
N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteases purified from Bothrops jararaca venom
Toxicon
(2002)
Chimeric derivative of fibrolase, a fibrinolytic enzyme from southern copperhead venom, possesses inhibitory activity on platelet aggregation
Arch. Biochem. Biophys.
Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake
Toxicon
A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization
Arch. Biochem. Biophys.
Cloning and functional expression of the mucrosobin protein, a β-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)
Protein Expr. Purif.
Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting in a marine green alga, Codium divaricatum
Comp. Biochem. Physiol., Part B Biochem. Mol. Biol.
Purification and characterization of two fibrinolytic enzymes from marine green alga, Codium intricatum
Comp. Biochem. Physiol., Part B Biochem. Mol. Biol.
A fibrinolytic enzyme from a marine green algae, Codium latum
Phytochemistry
Fibrinolytic and anticoagulative activities from the earthworm Eisenia foetida
Comp. Biochem. Physiol., Part B Biochem. Mol. Biol.
Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (Chinese Mantis)
Biochem. Biophys. Acta
Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese Mantis)
Insect Biochem. Mol. Biol.
Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidoptera, Saturniidae)
Toxicon
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2021, Toxicology LettersCitation Excerpt :Additionally, using rats as an experimental model, we also found increased plasmin activity in plasma earlier in the envenomation (between 2 h and 6 h after LOBE injection s.c.) and increased urokinase activity after 24 h of venom injection (Berger et al., 2010b). Until now no direct plasminogen activating activity has been found in crude L. obliqua venom, even though a fibrinolytic enzyme named lonofibrase has been isolated from hemolymph, and sequences of serine proteinases with homology to fibrinolytic enzymes were detected in hemolymph and bristle extracts (Pinto et al., 2004; Veiga et al., 2005). Taken together, all these data highlight the importance of fibrinolysis activation during L. obliqua envenomation.
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