The venom of the Lonomia caterpillar: An overview

ARTICLE IN PRESS

Toxicon 49 (2007) 741–757 www.elsevier.com/locate/toxicon

Review

The venom of the Lonomia caterpillar: An overview Linda Christian Carrijo-Carvalho, Ana Marisa Chudzinski-Tavassi Laboratory of Biochemistry and Biophysics, Butantan Institute, Av. Vital Brazil 1500, Sa˜o Paulo 05503-900, SP, Brazil Received 3 February 2006; received in revised form 17 November 2006; accepted 24 November 2006 Available online 10 January 2007

Abstract Contact with the Lonomia caterpillar causes numerous accidents, especially in Venezuela and the southern region of Brazil, where it is considered a public health problem. The Lonomia obliqua venom causes disseminated intravascular coagulation and a consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The venom of Lonomia achelous also causes hemorrhage, but through increased fibrinolysis. In vivo and in vitro studies have shown that the venom of the Lonomia caterpillar contains several toxins with procoagulant, anticoagulant and antithrombotic activities. These toxins also affect the endothelium. The recent construction of cDNA libraries of the transcripts from L. obliqua bristles enables the use of biotechnological approaches to study the venom. This paper presents an overview of the biochemical and biological properties of Lonomia caterpillar venom, discussing aspects of human accidents, experimental envenomation, toxins and targets and future perspectives. r 2007 Elsevier Ltd. All rights reserved. Keywords: Lepidoptera; Caterpillar; Hemorrhagic syndrome; Coagulation; Fibrinolyis; Lonomia

Contents 0. 1. 2. 3. 4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aspects of human envenomation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The poison apparatus of the Lonomia caterpillar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo studies with Lonomia crude venom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Experimental envenomation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Antithrombotic effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Median lethal dose and fatal complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Intravascular hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Nociceptive and edematogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro studies with Lonomia crude venom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Procoagulant activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Effect on the fibrinolytic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. achelous toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +55 11 3726 7222x2109; fax: +55 11 3726 1024.

E-mail address: [email protected] (A.M. Chudzinski-Tavassi). 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.11.033

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7.

8.

6.1. Prothrombin activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Factor V activators and inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Fibrinolytic proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. FXIII protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Extracellular matrix protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. obliqua toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Fibrinogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Hemolytic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Hyaluronidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. L. obliqua stuart factor activator (Losac). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. L. obliqua prothrombin activator protease (Lopap) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. L. obliqua toxins active in endothelial cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0. Introduction Caterpillars are the larval stage of moths and butterflies (Order: Lepidoptera), and are found in a variety of ecosystems worldwide (Balit et al., 2003). Some caterpillars cause problems in humans such as itching, others cause serious health problems, and yet others are agricultural pests. Caterpillar species that are a potential hazard for humans are found in about 12 families in the Lepidoptera order (Diaz, 2005). Accidental contact with the caterpillar’s bristles induces symptoms that range from mild cutaneous reactions to severe systemic reactions, depending on the species involved, the severity of the contact, and the physical conditions of the victim, e.g. health, age, and body weight. Sometimes contact with airborne hairs or with a dead caterpillar is sufficient to cause adverse reactions (Maier et al., 2003; Balit et al., 2001). Common symptoms include a burning sensation, urticating dermatitis and skin lesions (Vega et al., 2004), allergy (Vega et al., 1999; Aparicio et al., 2004), ocular injuries (Sood et al., 2004), osteochondritis (Dezhou, 1991), hemostatic disturbances, and renal and cerebral damages (Kelen et al., 1995). Diaz (2005) evaluated the global epidemiology of caterpillar envenoming and distinguished five distinct clinical syndromes: (1) erucism (local dermatitis and urticaria); (2) lepidopterism (generalized urticaria and other systemic symptoms); (3) dendrolimiasis (inflammatory polyarthritis and polychondritis and chronic osteoarthritis); (4) ophtalmia nodosa (conjunctivitis and intraocular migration of urticating hairs) and (5) consumptive coagulopathy (imbalances in the coagulation and fibrinolytic systems, resulting in a hemorrhagic syndrome).

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Caterpillar venoms have not been studied as extensively as the venoms of snakes, scorpions, spiders, bees and wasps. Among the most studied caterpillar venoms are those of moths of the Thaumetopoea, Euproctis and Lonomia genera. Caterpillars of the genus Thaumetopoea (family Notodontidae), commonly known as processionary tree caterpillars or pine caterpillars, are found especially in Europe, but some species are also found in the United States, Asia and Africa (Diaz, 2005). The bristles are toxic, and the main active toxin is a histamine-liberating toxin of 28 kDa named thaumetopoein (Lamy et al., 1986). Contact with the bristles causes dermatitis, allergic reactions (Maier et al., 2003; Aparicio et al., 2004) and conjunctivitis (Bessler et al., 1987). Caterpillars of the genus Euproctis (family Lymantriidae) are worldwide distributed and are commonly known as the mistletoe browntail moth caterpillars (Diaz, 2005). Contact with the hairs of these caterpillars causes urticating dermatitis and allergic bronchitis (Balit et al., 2001). The venom of these caterpillars has been partially characterized and contains ester hydrolases, phospholipase and proteases (de Jong et al., 1982; Bleumink et al., 1982). Lonomia caterpillars (family: Saturniidae) are found in South America and their venom affects mainly the coagulation system. The adverse effects observed are diffuse bleeding, renal failure and cerebral damage, sometimes progressing to death (Kelen et al., 1995; Arocha-Pin˜ango and Guerrero, 2003). Accidents are most common on fruit trees close to rural residences, which can have clusters of the caterpillar (Correˆa et al., 2004; Diaz, 2005).

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Two species of Lonomia are commonly involved in human accidents. Lemaire (1972) distinguished the Brazilian caterpillar Lonomia obliqua (Walker) and the Venezuelan caterpillar Lonomia achelous (Cramer). The venoms of these species cause similar clinical effects, but the mechanism by which they cause their adverse effects seem different: L. obliqua venom has procoagulant activity (Donato et al., 1998; Reis et al., 1999) whereas L. achelous venom has both procoagulant and anticoagulant activity (Arocha-Pin˜ango and Guerrero, 2001). The main active toxins identified in the venom of L. achelous are lonomin II (Arocha-Pin˜ango and Guerrero, 2003), which has direct fibrinolytic activity, and lonomin V, which degrades coagulation factor XIII (Guerrero et al., 1997a, b). In L. obliqua bristle extract, two procoagulant toxins have been identified: a factor X activator named Losac (L. obliqua Stuart-factor activator) (Alvarez Flores et al., 2006) and a prothrombin activator named Lopap (L. obliqua prothrombin activator protease) (Reis et al., 2001a). Lopap seems to be an important factor in the hemorrhagic syndrome caused by contact with the L. obliqua caterpillar (Reis et al., 1999, 2001b). In recent years the study of biological properties of the venom of the Lonomia caterpillars and its mechanisms of action has accelerated. New approaches have been introduced, including cellular and molecular methods (Chudzinski-Tavassi and Alvarez Flores, 2005) and cDNA libraries (Chudzinski-Tavassi et al., 2004; Veiga et al., 2005). This paper reviews the information currently available about the venom of the Lonomia caterpillar, and correlates its biochemical properties with clinical data and experimental studies. 1. Aspects of human envenomation The clinical symptoms that follow L. obliqua envenomation are very similar to those observed after L. achelous envenomation (Arocha-Pin˜ango et al., 1992). These symptoms include local burning pain, erythema, edema, headache, nausea and vomiting (Kelen et al., 1995; Duarte et al., 1996; Fan et al., 1998; Zannin et al., 2003), hematomas, hematuria, ecchymosis (Duarte et al., 1996; Zannin et al., 2003; Correˆa et al., 2004), anemia and leucocytosis (Arocha-Pin˜ango and Guerrero, 2001). Hemorrhage occurs in the skin, mucosa and viscera (Kelen et al., 1995; Zannin et al., 2003; Caovilla and Barros, 2004).

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Potentially fatal complications include acute renal failure (Duarte et al., 1990; Burdmann et al., 1996) and intracerebral bleeding (Kelen et al., 1995; Burdmann et al., 1996; Duarte et al., 1996). Acute renal failure was observed in 2% of patients admitted after contact with L. obliqua (Gamborgi et al., 2006), and 10% of these developed chronic renal failure. The patients with acute renal failure presented higher frequency of hematuria and the larger changes in coagulation parameters. However, the pathogenesis of the renal complications is unknown. Hemodynamic changes, renal ischemia, a massive deposition of fibrin in the glomerular capillaries associated with disseminated intravascular coagulation, and direct actions of venom toxins may contribute to this pathogenesis (Duarte et al., 1994; Fan et al., 1998; Gamborgi et al., 2006). Renal insufficiency is more frequent after contact with L. obliqua than with L. achelous (Arocha-Pin˜ango and Guerrero, 2001). The pathophysiologic processes involved in the hemorrhagic syndrome in the patients envenomed after contact with L. obliqua and L. achelous bristles are not completely known. Arocha-Pin˜ango and coworkers suggested that the hemorrhagic syndrome resulting from contact with the L. achelous caterpillar is primarily caused by activation of fibrinolysis and a mild disseminated intravascular coagulation (Arocha-Pin˜ango, 1967; Arocha-Pin˜ango et al., 1992; Arocha-Pin˜ango and Guerrero, 2003). Affected patients may present prolonged prothrombin time, activated partial thromboplastin time and thrombin time (Table 1), and these parameters can be corrected by the addition of normal plasma. In addition, increased euglobulin lysis time in the patient’s plasma indicates a severe fibrinolysis. Plasma from these patients reduces the lysis time and increases the lysis area on fibrin plates (Arocha-Pin˜ango and Guerrero, 2003). Treatment after contact with L. achelous consists of administration of anti-fibrinolytic drugs such as e-amino caproic acid and aprotinin (Arocha-Pin˜ango and Guerrero, 2001). The only large-scale clinical study (105 patients) on L. obliqua envenomation to evaluate blood coagulation and fibrinolysis parameters showed prolonged clotting times, decreased levels of coagulation factors and enhanced levels of fibrin degradation products (Zannin et al., 2003, Table 1). Similar changes are observed in disseminated intravascular coagulation (Wada et al., 1999; Taylor et al., 2001; Levi et al., 2002), but whereas disseminated intravascular

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Table 1 Coagulation and fibrinolysis measurements from patients and laboratory animals envenomed by Lonomia caterpillars

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coagulation is accompanied by a reduced platelet count, L. obliqua poisoning is not, suggesting different mechanisms for the changes in coagulation. Fibrinogen levels fell in the majority of poisoned patients and the extent of fibrinogen loss was correlated with the severity of bleeding manifestations. The most prominent changes in hemostatic parameters were observed in patients presenting fibrinogen depletion and more severe coagulopathy (Zannin et al., 2003). All these data indicate that the hemorrhagic syndrome induced by L. obliqua venom is a consequence of systemic intravascular activation of coagulation, leading to a consumptive coagulopathy and secondary activation of fibrinolysis. 2. Epidemiology The genus Lonomia includes 26 species found on the American Continent (Lemaire, 1972). However, only L. obliqua and L. achelous cause severe accidents, leading to hemorrhagic syndrome (Arocha-Pin˜ango et al., 1992; Kelen et al., 1995; Duarte et al., 1996; Fan et al., 1998; ArochaPin˜ango and Guerrero, 1999; Zannin et al., 2003). L. achelous is found especially in Venezuela (Arocha-Pin˜ango et al., 1992) and French Guyana (Couppie et al., 1998). A brown variety of L. achelous is found in the north of Brazil, where it is classified as L. diabolous. L. obliqua is found in the south of Brazil in the States of Rio Grande do Sul, Santa Catarina and Parana´ (Kelen et al., 1995; Rubio, 2001). The species appears to be spreading to the southeast of Brazil, and recent accidents with the species were reported in the states of Sa˜o Paulo (Fan et al., 1998), Rio de Janeiro (Correˆa et al., 2004) and Minas Gerais (informal publications). L. obliqua is also found in Uruguay, Paraguay and Argentina (Lemaire, 1972; De Roodt et al., 2000). Since 1989 the number of human accidents caused by L. obliqua caterpillars has been increasing in the southern region of Brazil (Duarte et al., 1990; Kelen et al., 1995; Rubio, 2001; Zannin et al., 2003; Diaz, 2005). Most victims were male (63%), many were between 0 and 19 years old (45%), and lesions are especially common on the hands (38%). The reported death rate is 2.5% (Rubio, 2001). An antiserum is produced by the Butantan Institute in Sa˜o Paulo, Brazil. It effectively reverses the coagulation disorders induced by L. obliqua venom (Dias da Silva et al., 1996; Rocha-Campos et al., 2001), and patients treated with this antiserum recover rapidly (Caovilla and Barros, 2004).

Fig. 1. Lonomia obliqua caterpillar.

3. The poison apparatus of the Lonomia caterpillar The Lonomia caterpillar has brownish-green bristles of different sizes on the body (Fig. 1), and these structures are present on all larval stages, from the first to the sixth larval instars (Lorini and Corseul, 2001). The bristles are formed by a nonporous tegument from the caterpillar cuticle and have a hollow canal that stores the venom (Veiga et al., 2001). The distal end of the bristle has a dark, thin, chitin-rich tip that easily breaks, releasing the venom from the internal canal. The venom is produced by a secretory epithelium above the tegument, both tissues outgrowths as a continuous evagination of the body to form the bristles. The venomous spicules and spines of moth caterpillars contain non-proteic and proteic components (Kawamoto and Kumada, 1984). Lonomia venom is mainly composed of proteins (Kelen et al., 1995; Arocha-Pin˜ango et al., 2000; Rocha-Campos et al., 2001) and contains serine proteases (Donato et al., 1998; Reis et al., 1999; Chudzinski-Tavassi and Alvarez Flores, 2005) and probably glycoconjugates (Veiga et al., 2001), among others. It has been proposed that the bristle extract may share similarities with the hemolymph (Veiga et al., 2001; Diaz, 2005) since the structures involved in the production of venom are located in the proximity of the body fluids. Thus, proteins identified in the bristle extract may play a role in processes related to the moth development such as regulation of the cell cycle. 4. In vivo studies with Lonomia crude venom 4.1. Experimental envenomation Various in vivo studies have been carried out to aid understanding of the biological mechanisms triggered by Lonomia caterpillar venom (Marval

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et al., 1999; Rocha-Campos et al., 2001; Prezoto et al., 2002, Silva et al., 2004a). The coagulation disorders observed in humans after contact with Lonomia caterpillars can be at least partially reproduced in experimental animals (see Table 1). Administration of the crude extract of L. obliqua bristles to rats, rabbits, and mice causes a dosedependent increase of clotting time (prothrombin time, activated partial thromboplastin time and whole blood clotting time) and bleeding time, and can render the blood unable to coagulate (Kelen et al., 1995; Prezoto et al., 2002; Reis et al., 2001b). Bristle extract also reduced plasma levels of fibrinogen and factor XIII and increased fibrin degradation products (Table 1). Factor XIII is a transglutaminase that stabilizes the clot by covalent cross-linking fibrin (Davie, 1995) and its inactivation would result in the impairment of clot formation and hemorrhage. As indicated in Table 1, the low factor XIII levels observed in laboratory animals (Prezoto et al., 2002; Fritzen et al., 2003) and humans (Zannin et al., 2003) envenomed by L. obliqua may be secondary to activation of clotting. In vitro studies showed that L. obliqua venom does not change factor XIII levels in fibrinogen-depleted plasma (Fritzen et al., 2003). Similar experiments show that L. achelous venom causes a dose-dependent decrease of fibrinogen, plasminogen and factor XIII (Table 1), but does not alter clotting time and fibrin plate lysis tests in laboratory animals (Marval et al., 1999). L. achelous venom directly degrades factor XIII (Guerrero et al., 1997b), whereas L. obliqua venom lacks factor XIII degradation activity (Fritzen et al., 2003). 4.2. Antithrombotic effect Since Lonomia caterpillar venom acts as an anticoagulant in vivo, experimental thrombosis studies were carried out to investigate whether the venom has antithrombotic activity. Prezoto et al. (2002) showed that doses of L. obliqua venom that do not cause spontaneous bleeding prevent the formation of thrombi, but do not dissolve already formed thrombi. These results are in agreement with the finding that L. obliqua venom does not contain enzymes capable of degrading cross-linked fibrin in vitro (Fritzen et al., 2003). The anticoagulant effect of L. obliqua venom is probably caused by depletion of fibrinogen as a consequence of consumptive coagulopathy.

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Unlike L. obliqua venom, L. achelous venom has thrombolytic activity (Guerrero et al., 2001). The lonomin V fraction from L. achelous venom not only inhibits thrombus growth but also induces lysis of preformed thrombi. These data reinforce the evidence that the venoms of L. obliqua and L. achelous trigger different mechanisms of the coagulation system. L. achelous venom predominantly activates fibrinolysis, whereas L. obliqua venom predominantly activates blood coagulation. 4.3. Median lethal dose and fatal complications The lethal dose (LD50) of L. obliqua venom in mice is about 10 mg/kg body weight (RochaCampos et al., 2001). Even large doses are not fatal to rats (Dias da Silva et al., 1996). There are indications that venom-treated animals develop the renal complications and intracerebral bleeding seen in human patients. These complications may be due, at least partly, to damage in these tissues, since the venom causes damage to the blood brain barrier (Silva et al., 2004a). The mechanism by which this damage occurs and the venom components responsible are still unclear. Silva et al. (2004b) showed that the venom was present in the kidney and liver, using immunohistochemistry (peroxidase-labeled rabbit antiserum raised against L. obliqua venom), but could not detect the venom in the brain. After intraperitoneal injection of 125 I-labeled L. obliqua venom in mice, the label was detected in kidney, urine, liver, lung, spleen and blood (Rocha-Campos et al., 2001). The highest levels were found 1 h after injection in the blood and kidneys, and the venom was almost completely cleared by 24 h. These findings are compatible with evidence that circulating blood clotting factors and possibly the vascular endothelium are mains target of the venom (Kelen et al., 1995; Fritzen et al., 2005; Alvarez Flores et al., 2006). The biodistribution of L. achelous venom is not known. 4.4. Intravascular hemolysis Seibert et al. (2004) showed that L. obliqua venom causes intravascular hemolysis in rats. This complication however seems rare in humans. The two cases described so far showed reduced hemoglobin and platelet levels (Correˆa et al., 2004; Malaque et al., 2006). Since clinical reports typically show normal platelet counts in patients envenomed by L. obliqua (Zannin et al., 2003) this may be an atypical

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Table 2 Activities and toxins identified in Lonomia caterpillar venom Species

Activity/toxin

MW (Da)

Characteristics

In vivo effect

Reference

L. obliqua

Prothrombin activator (Lopap)

69,000 (tetramer)

Serine protease, Ca2+ increase its activity

Consumptive coagulopathy

Factor X activator (Losac)

43,000

Serine protease

—

Donato et al. (1998) and Reis et al. (2001a, b) Donato et al. (1998) and Flores et al. (2004)

Phospholipase A2-like

15,000

ab Fibrinogenase (Lonofibrase)

35,000

Ca-independent Indirect hemolytic activity in human and rats red blood cells Unable to affect crosslinked fibrin

Intravascular hemolysis and hemoglobinuria —

Hyaluronidases

49,000

Anti-apoptotic proteina

53,000 51,000

Nociceptive (NI)

—

Edematogenic (NI)

—

Prothrombin activator (Lonomin III)

L. achelous

Seibert et al. (2003,2004, 2006)

Displays hydrolase activity as a bendohexosaminidase. Degrades purified hyaluronic acid, purified chondroitin sulphate and extracellular matrix.

—

Fritzen et al. (2003), Veiga et al. (2003) and Pinto et al. (2004)a Gouveia et al. (2005)

Activity in Spodoptera frugiperda (Sf-9) cell culture In vivo effect inhibited by indomethacin pretreatment In vivo effect inhibited by Loratadine pretreatment

—

Souza et al. (2005)

Nociceptive response

Bastos et al. (2004)

Edematogenic response

Bastos et al. (2004)

—

Ca2+ independent activity

—

Factor Xa-like (Lonomin IV)

—

—

Factor V activator (Lonomin VI:a) Factor V inactivator (Lonomin VI:i) Urokinase-like (Lonomin I)

—

Ca2+, factor V and phospholipids increase its activity Metalloprotaese

—

Guerrero and Arocha-Pin˜ango (1992) Guerrero and Arocha-Pin˜ango (1992) Lo´pez et al. (2000)

—

Serine protease

—

Lo´pez et al. (2000)

16,000–18,000

Inhibited by various protease-class inhibitors

Arocha-Pin˜ango and Pepper (1981) and Marval et al. (1999)

22,400

Serine protease

Decrease in fibrinogen, plasminogen and increase in fibrinogen degradation products —

22,700

Fibrinogen degradation products are different from those generated by plasmin Serine protease impairs fibrin/fibrinogen crosslinking by FXIIIa

Plasmin-like (Achelase I and Achelase II)

FXIII inactivator (Lonomin V)

—

NI: toxin not identified yet. Lopap: Lonomia obliqua prothrombin activator protease. Losac: Lonomia obliqua Stuart-factor activator. a Toxin identified in L. obliqua hemolymph.

Lysis of preformed venous thrombi and decrease in fibrinogen, plasminogen, a2antiplasmin and factor XIII levels

Amarant et al. (1991)

Guerrero et al. (1997a, b) and Guerrero et al. (2001)

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occurrence. A phospholipase A2 (PLA2) activity in L. obliqua venom (Table 2) seems to be involved in the intravascular hemolysis seen in rats (Seibert et al., 2003). L. achelous venom also normally does not reduce platelet counts in humans (Arocha-Pin˜ango et al., 1992; Arocha-Pin˜ango and Guerrero, 2003), but a few cases of anemia have been reported (ArochaPin˜ango and Guerrero, 2001). The effects of envenomation in animal models may not reproduce exactly those seen in human victims. L. obliqua venom induced hemolysis and hemoglobinuria, but not hematuria in rats, whereas in humans hematuria can be observed (Kelen et al., 1995; Zannin et al., 2003). Rats also did not present the spontaneous bleeding observed in humans (Seibert et al., 2004). It is possible that some of these differences are due to differences in dose rather than species. 4.5. Nociceptive and edematogenic effects The local effects induced by L. obliqua venom have been studied in animal models. Injection of the crude venom in the hind paw of the rat causes edema and pain (Bastos et al., 2004). Edema and pain seem to be mediated by distinct factors in the venom, which have not been identified yet. The short-lived nociceptive response does not seem to be due to the substances causing the hemorrhagic syndrome, and is inhibited by pretreatment with the inhibitor of prostaglandin synthesis indomethacin. It seems likely that this response is caused by PLA2 activity in the venom. PLA2 can generate large amounts of arachidonic acid that can be converted to prostaglandin by cyclooxygenase. On the other hand, PLA2 activity seems to have little role in edema, since edema in the rat paw is not blocked by indomethacin (Bastos et al., 2004). These local effects appear similar to those elicited by the venom in humans. 5. In vitro studies with Lonomia crude venom It is currently accepted that the main effect of Lonomia venom is on the haemostatic system. The effect of L. obliqua venom is mediated mainly by thrombin formation secondary to the action of procoagulant toxins at different levels of the coagulation cascade (Kelen et al., 1995; Donato et al., 1998; Reis et al., 2001a; Prezoto et al., 2002, Fritzen et al., 2003). On the other hand, L. achelous

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venom activates both fibrinolysis and clotting pathways. 5.1. Procoagulant activity Kelen et al. (1995) were the first to examine the effect of L. obliqua venom on hemostasis in vitro and demonstrated that the crude extract induces a dose-dependent procoagulant activity that is increased by calcium ions. Donato et al. (1998) showed that L. obliqua venom induces clot formation by triggering activation of both prothrombin and factor X, indicating the presence of a factor X activator and a calcium-dependent prothrombin activator. The factor X activator Losac (L. obliqua Stuart-factor activator) and the prothrombin activator Lopap (L. obliqua prothrombin activator protease) were later purified and characterized (Alvarez Flores et al., 2006; Reis et al., 2001a). These two toxins are discussed in Sections 7.4 and 7.5. Prothrombin activation may be critical for the comsumptive coagulopathy induced by L. obliqua venom. The crude extract causes a dramatic reduction in clotting time in recalcified citrated human plasma depleted of factor V or factor X (Donato et al., 1998). In addition, the L. obliqua venom fails to clot purified fibrinogen (Kelen et al., 1995; Donato et al., 1998; Fritzen et al., 2003) and to hydrolyze chromogenic substrate specific for thrombin (Donato et al., 1998). The venom also does not induce platelet aggregation in vitro (Donato et al., 1998; Chudzinski-Tavassi et al., 2001). L. achelous crude venom displays a direct prothrombin activator activity, which activates purified prothrombin in the absence of phospholipids and calcium, and a factor Xa-like activity (Arocha-Pin˜ango et al., 2000). In addition, the crude venom has a factor V activator activity that stimulates procoagulant activity (Lo´pez et al., 2000). However, this venom does not present thrombin-like activity since it failed to clot purified fibrinogen or hydrolyze S-2238, a chromogenic substrate specific for thrombin. 5.2. Effect on the fibrinolytic system L. achelous venom has fibrinolytic activity on fibrin plates, has urokinase-like activity on the chromogenic substrate S-2444, and induces whole blood clot lysis. Both a direct plasmin-like, and a

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plasminogen-dependent fibrinolytic activity contribute to the fibrinolytic activity of the whole venom (Arocha-Pin˜ango et al., 2000). In vitro studies confirmed clinical data (Zannin et al., 2003) and in vivo studies (Prezoto et al., 2002) that the venom of L. obliqua, in contrast that of L. achelous, is little effective at activating the fibrinolytic system (Kelen et al., 1995; Fritzen et al., 2003). L. obliqua venom does not activate plasminogen, and fails to lyze fibrin plates even after prolonged incubation (Fritzen et al., 2003). L. obliqua venom fails to degrade purified factor XIII and to form a complex with this coagulation factor, as shown by gel filtration and SDS-PAGE (Fritzen et al., 2003). The dansylcadaverin method failed to detect factor XIII inhibitory activity in the venom (Fritzen et al., 2003). Thus, all evidence indicates that fibrinolysis in patients envenomed by L. obliqua is not caused by a direct action of the venom on the fibrinolytic system, but is secondary to disseminated intravascular coagulation.

called fraction I) has FXa-like activity, since its activity is increased in the presence of factor V, phospholipids and calcium. Lonomin IV is inhibited by a protease inhibitor in the same mode as FXa (Arocha-Pin˜ango et al., 2000).

6. L. achelous toxins

L. achelous venom fractions with fibrinolytic activity can lyze whole blood clots and fibrin plates and their activity is not inhibited by human plasma, suggesting they are different from human endogenous fibrinolytic compounds. Plasminogen-dependent fibrinolytic toxin has urokinase plasminogen activator (uPA)-like activity. This toxin was named Lonomin I and has an apparent molecular mass of 16–18 kDa and a pI410. Lonomin I is stable over a wide range of pH values and temperatures and is inhibited by various protease inhibitors (Arocha-Pin˜ango and Pepper, 1981; Arocha-Pin˜ango et al., 2000). The plasmin-like toxins isolated from L. achelous venom are a 22.4 kDa-toxin called Achelase I and a 22.7 kDa-toxin called Achelase II. These enzymes showed a pI of 10.5 and 8.5, respectively (Amarant et al., 1991). Although they have a direct fibrinolytic action independently of plasminogen, the fibrinogen degradation products generated by the proteolytic activity of these enzymes are different from those generated by the action of plasmin on fibrinogen. The amino acid sequences of Achelase I and II showed high similarity with lepdopteran thrombinand trypsin-like serine proteases (Arocha-Pin˜ango et al., 2000).

The toxins and activities identified in Lonomia venom are summarized in Table 2. The hemolymph of L. achelous contains several fibrinolytic compounds: Achelase I and Achelase II (Lonomin II) have plasmin-like activity (Amarant et al., 1991), and Lonomin V degrades fibrin, fibrinogen and human factor XIII, impairing fibrin cross-linking (Guerrero et al., 1997a, b). Lonomin V also has urokinase-like activity that activates plasminogen (Arocha-Pin˜ango and Pepper, 1981; Arocha-Pin˜ango et al., 2000). Two types of prothrombin activators are present in L. achelous hemolymph: a calcium-independent compound (Lonomin III) and a factor Xa-like activity (Lonomin IV) (Guerrero and Arocha-Pin˜ango, 1992). A factor V activator (Lonomin VI:a) and a factor V inhibitor (Lonomin VI:i) (Guerrero and Arocha-Pin˜ango, 1989; Lo´pez et al., 2000) have been also described. The venom also contains Lonomin VII, a toxin with kallikreinlike activity (Arocha-Pin˜ango and Guerrero, 2001). 6.1. Prothrombin activators L. achelous venom contains two prothrombin activators (Guerrero and Arocha-Pin˜ango, 1992). Lonomin III directly activates prothrombin independently of the prothrombinase complex (phospholipids and calcium ions). Lonomin IV (also

6.2. Factor V activators and inhibitors The action of the direct FV activator Lonomin VI:a on FV seems to be similar to that of plasmin and thrombin (Lo´pez et al., 2000; Arocha-Pin˜ango and Guerrero, 2001). Lonomin VI:a is thermostable, has maximal activity at acid pH, and is inhibited by metalloprotease inhibitors, indicating that it is a metalloproteinase. The venom also contains the FV inactivator Lonomin VI:i (ArochaPin˜ango and Guerrero, 2001). Lonomin VI:i is thermolabile, has maximal activity at basic pH, and has serine protease-like activity (Lo´pez et al., 2000). 6.3. Fibrinolytic proteases

6.4. FXIII protease Lonomin V causes a dose-dependent degradation of factor XIII/XIIIa, with the generation of peptide

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fragments of low molecular weight and concomitant loss of FXIII transglutaminase activity (Guerrero et al., 1997b). Consequently, Lonomin V impairs fibrin/fibrinogen cross-linking by FXIIIa (Guerrero et al., 1997a). Lonomin V activity is stable over a wide range of pH values and temperatures, but is completely inhibited by serine protease inhibitors such as diisopropylfluorophosphate (DFP) and phenylmethylsulphonyl fluoride (PMSF), and by iodoacetamide, which blocks active site cysteine. This inhibition profile suggests that Lonomin V is a serine protease with a free cysteine residue that is essential for the enzymatic activity (Guerrero et al., 1999). Lonomin V seems to be identical to Lonomin I and this toxin has been called Lonomin I/V (Arocha-Pin˜ango and Guerrero, 2001). N-terminal sequence analysis of this toxin showed a homology of 75% with plasmin-like toxins from L. achelous (Arocha-Pin˜ango and Guerrero, 2003).

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presence of sequences related to trypsin-like enzymes, blood coagulation factors, prophenoloxidase cascade activators, cysteine proteases, phospholipase A2, serpins, cystatins, antibacterial proteins and lipocalins, and others (GenBank accession number: AY829732–AY829859; Veiga et al. 2005). The most abundant toxin was a lipocalin with an apparent molecular weight of approximately 20 kDa, and analysis of its N-terminal sequence shows 100% homology with Lopap (GenPept accession number: AAW88441). These data corroborate the previous studies discussed earlier, and again point out Lopap as one of the most important toxins from L. obliqua venom. L. obliqua bristle extract contains various proteic toxins, as demonstrated by the protein bands recognized by antilonomic serum on SDS-PAGE (Dias-da-Silva et al., 1996; Rocha-Campos et al., 2001). The proteins corresponding to each biological activity seen have not yet been identified.

6.5. Extracellular matrix protease 7.1. Fibrinogenases Lucena et al. (2006) recently reported serine protease-like toxins in L. achelous hemolymph that degrade extracellular matrix proteins such as laminin, vitronectin and fibronectin. These toxins were identified as chromatographic fractions FDII, Lonomin V and Lonomin V-2, have an apparent molecular mass of 25 kDa, and show activity similar to plasmin and urokinase. These toxins may contribute to the hemorrhagic events triggered by L. achelous venom because they may facilitate the spreading of the venom through the victim’s body and aggravate hemorrhage by destroying the capillaries. 7. L. obliqua toxins A cDNA cloning library of the transcripts from L. obliqua bristles was constructed and several clones were sequenced (http://www.ncbi.nlm.nih. gov) in 2004. The sequences were assembled in 702 genes that encoded lipocalins, hemolins, serine proteases, serine protease inhibitors, serpins, tumor suppressors, ribosomal, structural and cell cycle proteins (GenBank accession number: CX815710– CX817210, CX820335–CX820336, AY908986; Chudzinski-Tavassi et al., 2004). Lopap (GenBank accession number: AY908986) accounted for 1.6% of the total clones. More recent cDNA libraries of the bristles and the integument of L. obliqua similarly revealed the

Despite all data evidence demonstrating that fibrinolysis activation is not a primary action of L. obliqua venom, some in vitro studies showed the presence of fibrinogenolytic activity in this venom (Veiga et al., 2003; Fritzen et al., 2003). Pinto et al. (2004) described a 35-kDa fibrinogenolytic protein named Lonofibrase that was purified from L. obliqua hemolymph. This enzyme cleaves preferentially Aa chain fibrinogen, and is less effective against the Bb chain. However, it has been demonstrated that fibrinogen hydrolysis is weak, even with high concentrations of the L. obliqua crude venom (at 1:2 stoichiometry) and after prolonged incubation (Fritzen et al., 2003). The venom increased the fibrinogen clotting time, but the fibrin clot was not lyzed even after 24 h. Thus, the fibrinogenolytic activity fails to affect cross-linked fibrin and may be irrelevant in thrombus lysis. 7.2. Hemolytic factors L. obliqua venom has direct and indirect hemolytic activity on rat erythrocytes in vitro (Seibert et al., 2003). The direct effect was observed only with higher venom concentrations. A later study shows that L. obliqua venom also induces hemolysis in rats (Seibert et al., 2004). The indirect effect is due to a phospholipase A2 (PLA2)-like toxin (Seibert et al.,

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2003). The purified PLA2 is a thermolabile enzyme of 15 kDa, it has a pI of 5.9, it is calcium dependent, and its maximum activity is at pH 8.0. Its N-terminal sequence suggests that this toxin is a group III PLA2 (Seibert et al., 2006). 7.3. Hyaluronidases Gouveia et al. (2005) described two hyaluronidases in L. obliqua venom. The enzymes were called Lonoglyases. These type b endohexosaminidase hydrolases have apparent molecular masses of 49 and 53 kDa and optimal activity at pH 6.0–7.0. They degrade hyaluronic acid and purified chondroitin sulphate, but not heparan sulphate or dermatan sulphate. Hyaluronidases probably contribute to the local effects of the venom. They may also facilitate the passage of venom through the dermis. The L. obliqua venom hyaluronidases may be of great interest, since they might affect cancer cell growth and tumor invasion, and serve as a tool in cell biology studies (Csoka et al., 2001; Matsushita and Okabi, 2001) and for pharmaceutical applications (Smith et al., 1997; Menzel and Farr, 1998). 7.4. L. obliqua stuart factor activator (Losac) Losac is the first, and so far only, factor X activator purified from L. obliqua venom. It is a single polypeptide chain protein of about 43 kDa (Chudzinski-Tavassi and Alvarez Flores, 2005; Alvarez Flores et al., 2006). This enzyme activates factor X in a concentration-dependent manner, and the FXa formed is able to integrate the prothrombinase complex. Losac activity was totally inhibited by PMSF, indicating that this enzyme is a serinelike protease (Chudzinski-Tavassi and Alvarez Flores, 2005). The partial amino acid sequence of Losac showed no similarity with other factor X activator sequences (Flores et al., 2004). The biochemical properties of Losac are currently under investigation. 7.5. L. obliqua prothrombin activator protease (Lopap) The prothrombin activator Lopap is a 69-kDa tetrameric protein. This enzyme is the most studied L. obliqua toxin. It activates prothrombin in a dosedependent manner, generating thrombin able to clot purified fibrinogen (Reis et al., 1999, 2001a) and

reduce the clotting time (recalcification time) of citrated plasma (Reis et al., 2001a). The hydrolytic activity of Lopap is independent of the prothrombinase complex components, and is increased by calcium ions (Reis et al., 2001a). Although the prothrombinase complex is the physiological activator of prothrombin; prothrombin can also be activated by exogenous sources such as snake venom components (Rosing and Tans, 1992; Joseph and Kini, 2001). Prothrombin activators from snake venoms are grouped in several classes, according to their biochemical and enzymatic properties (Kini, 2005), but Lopap does not fit in any of the classes of snake prothrombin activators. The activity of Lopap is inhibited by antilonomic serum (Dias da Silva et al., 1996) and by serine protease inhibitors such as PMSF (Reis et al., 2001a). The mechanism of action of Lopap is similar to that of factor Xa in absence of prothrombinase components. Lopap cleaves prothrombin into products with molecular weights similar to prethrombin 2, fragments 1+2, and thrombin (Mann, 1994). Both Lopap and factor Xa induce the formation of prethrombin 2 by the cleavage of Arg284–Thr285 bond of prothrombin. Both subsequently form thrombin by the cleavage of the Arg320-Ile321 peptide bond. Lopap does not seem to form meizothrombin (Reis et al., 2001a, 2006). The thrombin generated by Lopap activates platelets in the same way as the a-thrombin formed by factor Xa. The amidolytic activity of thrombin generated by incubation of prothrombin with Lopap in a purified system is inhibited by antithrombin, a coagulation inhibitor that binds to a-thrombin (Chudzinski-Tavassi et al., 2001). However, it is not yet known if this thrombin behaves the same as that generated in vivo by the prothrombinase complex. Intravenous administration of purified Lopap to rats reproduced the hemorrhagic syndrome seen in human patients (Reis et al., 2001b), suggesting that Lopap plays an important role in L. obliqua envenomation. Depending on the dose administered, Lopap can trigger a dose-dependent inhibition of coagulation, probably due to fibrinogen depletion, rather than to platelet depletion (Reis et al., 2001b). In vitro studies demonstrated that Lopap does not have a direct effect on platelet function, since Lopap did not affect platelet aggregation induced by various agonists (Chudzinski-Tavassi et al., 2001).

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However, Lopap may have a dual indirect effect on platelets. On one hand, Lopap may induce a platelet aggregation by the generation of thrombin (Reis et al., 2001b). On the other hand, Lopap may inhibit platelet aggregation by inducing the release of platelet aggregation inhibitors from endothelial cells, such as nitric oxide (NO) and PGI2 (Chudzinski-Tavassi et al., 2001). The partial amino acid sequence from native Lopap was obtained (Reis et al., 2001a), and a recombinant monomeric form of this protein (rLopap) of 21 kDa was produced in Escherichia coli (Chudzinski-Tavassi et al., 2004; Reis et al., 2006). A protein from L. obliqua bristles presenting factor Xa-like activity was reported (Lilla et al., 2005), and its amino acid sequence demonstrated to be the same of that of Lopap. The Lopap sequence showed no similarity with the sequences of other prothrombin activators (Reis et al., 2001a), but is related to insecticyanin, a blue biliprotein isolated from the hemolymph of the tobacco hornworm Manduca sexta (Riley et al., 1984) and to biliverdinbinding protein-1 (BBP-1), an insecticyanin-type protein from the Eri-silkworm Samia Cynthia ricini (Saito, 1998). Insecticyanin, BBP-1 and Lopap are members of the lipocalin protein family. The lipocalins are small secreted proteins with high affinity for hydrophobic molecules (Flower et al., 1993). These proteins have a variety of functions including transport of retinol and pheromone, cryptic coloration, olfaction and enzymatic activities (Flower, 1996; Hieber et al., 2000; Irikura et al., 2003). In addition, lipocalin proteins can bind to specific cell-surface receptors and play a role in the regulation of immune response and cell viability, growth and differentiation (Flower, 1996). Therefore it seems possible that Lopap, in addition to its role as a poison in defense against predators, has a function in other vital processes within the caterpillar. 7.6. L. obliqua toxins active in endothelial cell cultures The endothelial surface can modulate coagulation and inflammation through several pathways (Vallet and Wiel, 2001; Cook-Mills and Deem, 2005). Therefore we investigated the effects of Lopap on the expression of mediators involved in coagulation, fibrinolysis and inflammation in cultured endothelial cells (HUVEC). Lopap stimulates the release of nitric oxide, a vasodilator and a potent inhibitor of

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platelet activation, and of prostacyclin (PGI2) (Fritzen et al., 2005). In addition, Lopap increases the expression of interleukin 8 (IL-8) and the cell adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and E-selectin (Chudzinski-Tavassi et al., 2001). Lopap failed to stimulate the endothelial release and synthesis of the prothrombotic factors tissue factor (TF) and von Willebrand factor (vWF), and the fibrinolytic factor tissue plasminogen activator (t-PA) (Chudzinski-Tavassi et al., 2001). Another interesting endothelial effect of Lopap is the inhibition of cell death (Fritzen et al., 2005). This effect is probably mediated by the liberation of NO, since NO acts as an endothelial survival factor, inhibiting apoptosis (Dimmeler and Zeiher, 1999; Rossig et al., 1999). An additional anti-apoptotic protein was recently identified in L. obliqua hemolymph (Maranga et al., 2003; Souza et al., 2005). This protein has an apparent molecular mass of 51 kDa and prevents apoptosis induced by nutrient depletion in insect cell cultures (Sf-9 cell lines, Souza et al., 2005). The factor X activator Losac also affects cell survival mechanisms. Losac inhibits apoptosis and stimulates proliferation in HUVEC (Alvarez Flores et al., 2006). This effect may depend on increased concentrations of NO. Losac also stimulates the production of the fibrinolytic factor tissue t-PA, which is involved in matrix remodeling (Kucharewicz et al., 2003). On the other hand, the expression of other molecules involved in inflammation and coagulation such as ICAM-1, PGI2, decay accelerating factor (DAF), IL-8, vWF and tissular factor were not affected by Losac (Alvarez Flores et al., 2006). 8. Concluding remarks The results presented in this review strengthen the notion that L. obliqua and L. achelous venoms have different components and that the pathways by which they cause a hemorrhagic syndrome may be different. L. obliqua causes the activation of factor X and prothrombin, resulting in a consumptive coagulopathy and consequent blood incoagulability. There are no indications that L. obliqua venom is able to directly activate fibrinolytic pathways, rather, fibrinolysis appears to be secondary to intravascular fibrin formation, which in turn is a consequence of an enhanced generation of thrombin.

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Although the clinical picture in L. achelous envenomation is similar to that of L. obliqua, with impaired blood coagulation and bleeding, the principal mechanism triggered by L. achelous venom is fibrinolysis, probably due to direct and indirect fibrinolytic activities and FXIII inactivator activity. The severe fibrinolysis may mask the procoagulant activities also found in L. achelous venom. However, the studies with L. obliqua used bristle extract, and those with L. achelous hemolymph, and it is not known if this contributes to the differences observed in the effects of the venom of both species. A diversity of proteins has been identified in L. obliqua venom by biochemical methods and by molecular biology and bioinformatics. However, the biochemical and biological properties and the function of L. obliqua venom proteins are poorly understood. Studies on these subjects may supply new data on L. obliqua envenomation and point out new molecules interesting for basic and applied sciences. The data discussed here indicate that Lopap, a prothrombin activator isolated from L. obliqua crude bristle extract, is probably one of the main factors causing the consumptive coagulopathy and hemorrhagic syndrome in the victims envenomed by L. obliqua, since its administration to laboratory animals triggered a condition similar to that in human poisoning (Reis et al., 2001b). Recombinant Lopap has now been obtained (Reis et al., 2006). Studies are in progress to elucidate effects of rLopap in vitro and in vivo that could account for the mechanisms of action of this enzyme. New perspectives include the molecular cloning of other toxins identified in the transcripts, which may be involved in the envenomation syndrome or which may be interesting for therapeutic and biotechnological purposes, and the cross-linking of transcriptomic and proteomic analysis of the L. obliqua bristle extract, which was done by 2-D electrophoresis and mass spectrometry. Molecular approaches bring a large amount of informative and quantitative data, which are critical for comparative and prospective analysis. From the cDNA library, new toxins can be identified, and obtained at large amounts in a homogeneous form by molecular cloning. On the other hand, studies at a cellular level may clarify the mechanisms of action of Lonomia toxins and point out new targets for these molecules, opening perspectives for therapeutic and biotechnological use.

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