Lonofibrase, a novel α-fibrinogenase from Lonomia obliqua caterpillars

Abstract

Envenomation caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by a profound hemorrhagic disorder. In the present study, we describe the characterization of a fibrin(ogen)olytic factor (lonofibrase) isolated from a venomous secretion of the caterpillars. The crude extract showed a dose-dependent inhibitory effect in the rate of thrombin-induced fibrinogen clotting and produced fragmentation of fibrinogen. Isolation of the fibrin(ogen)olytic enzyme was achieved by combining ion exchange chromatography followed by gel filtration in a fast protein liquid chromatography (FPLC) system. A single 35-kDa band was identified and the isolated enzyme named lonofibrase. Lonofibrase rapidly degrades Aα and Bβ chains of fibrinogen, also being able to cleave fibrin in a distinct way from that observed with plasmin. The presence of lonofibrase with both fibrinogenolytic and fibrinolytic activities in L. obliqua secretion is coherent with the severe hemorrhagic clinical profile resulting from envenomation caused by these insects.

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.