Regular ArticleFibrinogenolytic and procoagulant activities in the hemorrhagic syndrome caused by Lonomia obliqua caterpillars
Introduction
In spite of the efficiency and redundancy of the hemostatic system developed by mammals, along the evolution, many other animals evolved physiological processes capable of interfering with the hemostatic mechanisms. Such evolutionary trait gave chance to the appearance of blood-feeding animals like leeches, ticks, triatomines, mosquitoes, and bats (for a review, see Ref. [1]); others, such as snakes and spiders, make use of anti-hemostatic compounds to obtain and digest their preys. Among the well-known anti-hemostatic principles produced by animals are hirudin [2], batroxobin [3], [4], jararhagin [4], [5], and bothrojaracin [6], [7].
More recently, Lonomia caterpillars became known for producing venomous secretions that deeply interfere with the hemostatic system, leading to a serious bleeding disorder in men [8], [9], [10]. Interestingly, in the case of Lonomia, which feeds on leaves, the anti-hemostatic mechanisms seem to play an important role in protection against predators, rather than being involved directly with feeding or digestion. Two species of the genus Lonomia are known to cause such hemorrhagic syndrome: Lonomia achelous, which occurs in Venezuela and Northern Brazil, and L. obliqua, with occurrence in Southern Brazil [8], [9], [10]. Accidents with the latter became an increasing health problem in the State of Rio Grande do Sul, Brazil, where the caterpillars can be found even in urban areas. In the last 5 years, 688 cases of such hemorrhagic syndrome were reported in that area, including 1 death [11].
We recently reported a detailed study on the ultrastructure and morphology of Lonomia caterpillars, showing that the external tegument that covers the body of L. obliqua is very complex, composed of several chitinous specializations, including large bristles, also known as spicules or scoli [12]. Envenoming occurs when a person touches or smashes the caterpillars, breaking the spicules and releasing the venomous secretions inside the victim's skin and adjacent blood vessels. Accidents with Lonomia cause pain and burning sensation at the site of contact, followed by weakness, headache, fever, and vomiting some hours later. Bleeding itself may develop after 24 h or even after 1 week and includes ecchymoses, hematomas, hematuria, bleeding from scars and mucous membranes, pulmonary and intracerebral bleeding, and eventually acute renal failure [10], [13], [14]. Laboratory analysis of blood from patients envenomed by Lonomia revealed normal platelet count, anemia, long prothrombin time, low levels of fibrinogen (Fg), plasminogen, factor V and factor XIII, and high levels of fibrinogen degradation products (FDP) [15], [16], [17].
In vivo studies in which L. achelous venom was injected in rabbits reproduced some of the clinical consequences of the envenoming observed in humans, such as a drop in fibrinogen and in factor XIII levels, an increase in FDP [18], and a thrombolytic effect [19]. Concerning L. obliqua, while in vivo studies with bristle extract (BE) reported both anti-thrombotic and thrombolytic actions [20], most in vitro studies on the venom of this species report only procoagulant agents: a prothrombin activator, named Lopap (L. obliqua prothrombin activator protease) [21], [22] and a Factor X activator [23].
As we can conclude, despite the fact that envenoming by both species lead to similar complex hemorrhagic symptoms, accompanied by disseminated intravascular coagulation (DIC) and by acute renal failure, data so far indicate the presence of a fibrinolytic agent—which clearly explains the bleeding disorder—only in the venom of L. achelous, while no study indicates a similar activity in the venom of L. obliqua. Moreover, it is claimed that Lopap is the sole molecule responsible for the hemorrhagic syndrome caused by the latter species; it would act by consuming all fibrinogen from plasma, leading to DIC and, secondarily, to hemorrhage [17], [21].
These differences found between the two venoms may be due to the materials used by the different groups. Whereas studies on L. achelous use hemolymph and bristle extract as sources of the active principles, most studies with L. obliqua use only bristle extract and purified Lopap. In the present study, we investigate the action of L. obliqua crude bristle extract upon some blood coagulation and fibrinolysis parameters. Our findings show the presence of a fibrin(ogen)olytic activity in crude bristle extract of L. obliqua.
Section snippets
Materials
Plasmin (P) and all chromogenic substrates were purchased from Chromogenix (Sweden). Human citrated plasma was provided by Hospital de Clı́nicas de Porto Alegre from a pool of healthy donors; prothrombin and thrombin were prepared in our laboratory from the same plasma, according to the method described in Ding and Xu [24]. Factor X-deficient plasma, obtained from a female patient whose plasma exhibited very low Factor X levels, was kindly provided by Prof. Dr. Israel Roisenberg from the
Caterpillars and bristle extract
Lonomia obliqua caterpillars were kindly provided by Centro de Informação Toxicológica (CIT) in Porto Alegre and by Corpo de Bombeiros in Erechim (both in the state of Rio Grande do Sul). Crude bristle extract was obtained based on Silva et al. [25]. Briefly, the scoli (bristles) were cut at the base, homogenized in water and centrifuged (9600×g for 20 min); the supernatant was stored at −20 °C until use.
Procoagulant activity
To analyze the effect of L. obliqua bristle extract upon clotting, human citrated plasma was incubated with the extract prior to the induction of coagulation with calcium. As clearly shown in Fig. 1, bristle extract of L. obliqua caterpillars displays a potent procoagulant effect capable of shortening the clotting time of human plasma, even in the absence of calcium (and presence of EDTA). While the control plasma (with no sample added) took over 5 min to initiate coagulation after the addition
Discussion
Biochemical studies with the hemolymph and bristle extract of L. achelous reported the presence of several agents: a fibrinolytic activity and a plasminogen activator [18], [19]; Lonomin V, a serine proteinase that degrades factor XIII [16]; a metalloproteinase that activates factor V and another proteinase that inactivates factor V [29]; and a calcium-independent prothrombin-activating component [30].
In contrast, studies with L. obliqua venom reported mainly procoagulant activities, including
Acknowledgements
We acknowledge Centro de Informação Toxicológica (CIT) in Porto Alegre and Corpo de Bombeiros in Erechim (state of Rio Grande do Sul) for providing L. obliqua caterpillars. We thank Prof. Dr. Israel Roisenberg for kindly providing normal and Factor X-deficient human plasma, and Markus Berger for technical support. This work was supported by fellowships and grants by the Brazilian agencies CNPq (MCT, Brası́lia, DF), CAPES (MEC, Brası́lia, DF) and FAPERGS (Porto Alegre, RS). We also thank Dr.
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