ARTICLE IN PRESS
Toxicon 49 (2007) 663–669 www.elsevier.com/locate/toxicon
Kallikrein–kinin system activation by Lonomia obliqua caterpillar bristles: Involvement in edema and hypotension responses to envenomation C.B. Bohrera,1, J. Reck Juniora,b,1, D. Fernandesc, R. Sordic, J.A. Guimara˜esa, J. Assreuyc, C. Termignonia,d, a
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil Faculdade de Veterina´ria, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil c Departamento de Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Santa Catarina (UFSC), Floriano´polis SC, Brazil d Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil b
Received 13 April 2006; received in revised form 11 November 2006; accepted 13 November 2006 Available online 21 November 2006
Abstract Lonomia obliqua envenomation induces an intense burning sensation at the site of contact and severe hemorrhage followed by edema and hypotension, and after few days death can occur usually due to acute renal failure. In order to understand more about the envenomation syndrome, the present study investigates the role played by kallikrein–kinin system (KKS) in edematogenic and hypotensive responses to the envenomation by L. obliqua. The incubation of L. obliqua caterpillar bristles extract (LOCBE) with plasma results in kallikrein activation, measured by cromogenic assay using the kallikrein synthetic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). It was also showed that LOCBE was able to release kinins from low-molecular weight kininogen (LMWK). Moreover, it was demonstrated that previous administration of a kallikrein inhibitor (aprotinin) or bradykinin B2 receptor antagonist (HOE-140) significantly reduces the edema and hypotension in response to LOCBE, using mouse paw edema bioassay and mean arterial blood pressure analysis, respectively. The results demonstrate a direct involvement of the KKS in the edema formation and in the fall of arterial pressure that occur in the L. obliqua envenomation syndrome. r 2006 Elsevier Ltd. All rights reserved. Keywords: Lonomia obliqua; Venom; Kallikrein; Bradykinin; Edema; Hypotension
Abbreviations: Bk, bradykinin; i.p., intraperitoneal; DIC, disseminated intravascular coagulation; S-2302, H-D-Pro-Phe-Arg-pNA; HMWK, high molecular weight kininogen; LMWK, low molecular weight kininogen; HOE-140; Icatibant; D-Arg-[Ilyp3, Thi5, D-Tic7, Oic8]-Bk; LOCBE; Lonomia obliqua caterpillar bristles extract; MAP, mean arterial pressure; PK, pre-kallikrein; HF, Hageman factor; KKS, kallikrein–kinin system. Corresponding author. Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul. Address: Av. Bento Gonc- alves, 9500, P.O. Box 15005, ZIP Code 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 51 33166082. fax: +55 51 33167309. E-mail address:
[email protected] (C. Termignoni). 1 These authors contributed equally to this work. 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.11.005
ARTICLE IN PRESS 664
C.B. Bohrer et al. / Toxicon 49 (2007) 663–669
1. Introduction An intense burning sensation is caused by contact with Lonomia obliqua bristles (Arocha-Pin˜ango and Guerrero, 2001; Veiga et al., 2001; Coˆrrea et al., 2004), accompanied by erythema and edema. Despite the well-described characterization of L. obliqua proteins involved in haemostasis disorders (Pinto et al., 2006), the mechanisms involved in the venom noxious effects associated with vascular and inflammatory changes are not fully understood. Prostaglandins role is significant, since indomethacin pretreatment strongly inhibits the inflammatory effect of the venom (de Castro Bastos et al., 2004). On the other hand, L. obliqua caterpillar bristles extract (LOCBE)-derived edematogenic response seems to be mediated by histamine, since loratadine reduced LOCBE edematogenic effect (de Castro Bastos et al., 2004). After the initial inflammatory response, the venom causes disseminated intravascular coagulation (DIC), hypotension, fibrinolysis and hemorrhage (Donato et al., 1998; Veiga et al., 2003). The so-called hemorrhagic syndrome is characterized by reduction in plasminogen, fibrinogen and pre-kallikrein levels (Zannin et al., 2003; Pinto et al., 2004). Kinins are biologically active peptides, which are enzymatically released from protein precursors, low- and high-molecular weight kininogen (LMWK and HMWK, respectively). In physiological conditions, bradykinin (Bk) release is triggered by plasma pre-kallikrein (PK) activation (Kaplan and Silverberg, 1987). The active enzyme is able to activate Hageman factor (HF), which in turn produces more active kallikrein. In some pathological conditions, such as endotoxemia, activation of this enzymatic cascade may spread in plasma (Herald et al., 2003). The massive kallikrein–kinin system (KKS) activation leads to an increase in vascular permeability and to DIC and hypotensive responses, producing deleterious effects (Kaplan and Silverberg, 1987). In this work, the function of KKS in the edematogenic and hypotensive responses caused by L. obliqua envenomation was studied. Also, experiments to clarify how LOCBE modulates KKS and induces kallikrein activation and kinin releasing were done. 2. Materials and methods 2.1. Animals Female Swiss mice (weighing 20–25 g), female guinea pigs (150–200 g) and male Wistar rats
(200–250 g) were housed in a temperature-controlled (21–25 1C, in a 12-h light/dark cycle) room, with free access to water and food. All procedures were in accordance with NIH Animal Care Guidelines. 2.2. Drugs Bk was purchased from Sigma-Aldrich (Saint Louis, USA). Captopril was kindly provided by Prof. Ana Bergold (Faculdade de Farma´cia— Universidade Federal do Rio Grande do Sul, Brazil). Trasysols (aprotinin, 1.4 mg/mL) was a kind gift of Bayer do Brasil S. A.s (Sa˜o Paulo, Brazil). Human plasma was obtained at the University Hospital (Hospital de Clı´ nicas de Porto Alegre—Universidade Federal do Rio Grande do Sul, Brazil). S-2302 (H-D-Pro-Phe-Arg-pNA) was purchased from Chromogenix (Milano, Italy). HK and LK were purified in our laboratory according to Pierce & Guimara˜es (1974). HOE 140 was kindly provided by Prof. Joa˜o Batista Calixto (Departamento de Farmacologia—Universidade Federal de Santa Catarina, Brazil). 2.3. Caterpillars and bristle extract L. obliqua caterpillars were collected in Rio Grande do Sul state, southern Brazil, and provided by Centro de Informac- a˜o Toxicolo´gica (CIT) of Porto Alegre. LOCBE was obtained by cutting bristles at the base, macerating with a mortar and pestle the material in deionized water and centrifuging at 9600g for 20 min. The supernatant was collected and stored at 20 1C until use. 2.4. Protein estimation Protein quantification was done using the QuantiTM BCA assay kit (Sigma, Saint Louis, USA) according to manufacture’s instructions (Brown, 1989). LOCBE quantities were expressed as mg of protein. 2.5. Chromogenic assay To determinate kallikrein activity in plasma pretreated with LOCBE, a chromogenic assay was performed using a 96-well microplate reader spectrophotometer (SpectraMaxs, Molecular Devices Co., Sunnyvale, CA, USA) equipped with a temperature controller and shaking accessories: 50 mL of diluted
ARTICLE IN PRESS C.B. Bohrer et al. / Toxicon 49 (2007) 663–669
human plasma (1:10) were preincubated for 10 min at 37 1C with 12 or 24 mg of LOCBE and 20 mM HEPES buffer pH 7.4 to complete 100 mL. Then, the substrate S-2302 (H-D-Pro-Phe-Arg-pNA) was added (0.2 mM final concentration). The incubation mixtures were maintained at 37 1C and the reaction was followed measuring the absorbance at 405 nm, at 9 s of interval. Statistical analysis was done taking into account the values (expressed as mean and 7SEM, n ¼ 8) of substrate hydrolysis (pmol/min). 2.6. Guinea pig ileum bioassay The distal ileum of six female guinea pigs (150–200 g) was removed and washed with Tyrode solution (Webster and Prado, 1970). One centimeter ileum segments were placed in a 10-mL organ bath containing Tyrode solution, supplied with atmospheric air, with atropine sulfate (0.7 mM) and prepared isotonically with a tension of 1 g. The preparation was allowed to rest for 30 min. Contraction in response to kinin agonists were registered with a force transducer (Harvard Apparatus, Holliston, MA, USA) on a one-channel chart recorder. Showed results are representative of six independent experiments. 2.7. Mice paw edema Mice were injected subcutaneous with 50 mL of phosphate buffer saline (PBS) in the left hind paw (control). The animals were separated into three groups (eight mice per each treatment group) and the right hind paw received LOCBE (35 mg), 30 min after administration of a bradykinin B2 receptor antagonist, HOE-140 (100 nmol/kg, i.p.); plasma kallikrein inhibitor, aprotinin (8 mg/kg, i.p.) or PBS. The paw volume was measured with a plethysmometer as previously described (Ferreira, 1979). The difference in the volume between the right and left hind paws was taken as paw edema and was expressed in mL. 2.8. Mean arterial blood pressure Male Wistar rats were anesthetized with ketamine and xylazine (i.p. 90 and 15 mg/kg, respectively, supplemented at 45- to 60-min intervals). Then, heparinized PE-20 and PE-50 polyethylene catheters were inserted, respectively, into the left femoral vein for drug injections and into the right carotid artery for measure of mean arterial pressure (MAP). To
665
prevent clotting, a bolus dose of heparin (300 I.U.) was injected immediately after the vein cannulation. Body temperature was monitored by a rectal thermometer and maintained at 36 1C71 1C by a thermal surgical table. Drugs were diluted in sterile Dulbecco’s phosphate-buffered saline (PBS, in mM 137 NaCl, 2.7 KCl, 1.5 KH 2 PO4, and 8.1 NaHPO4; pH 7.4). Blood pressure data were recorded (at a 10-s sampling rate) with a Digi-Med blood pressure analyzer system (Model 190) connected to a digimed system integrator (Model 200; Louisville, KY, USA) software. Thirty minutes after induced anesthesia, the animals were separated in two groups (12 mice per each treatment group), one group received a single intravenous dose of bradykinin (Bk, 3 nmol/kg) and the other LOCBE (3 mg/kg). After the return of blood pressure to normal values, each group was subdivided in two (six mice each), one subgroup received HOE 140 (100 nmol/kg, i.v.) and other aprotinin (Trasylols, 4 mg/kg, i.v.). Then LOCBE or Bk was reinjected in all animals. 2.9. Statistical analysis Data are expressed as mean 7SEM of n animals. Statistical significance of mice paw edema assay and of chromogenic assay was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni or Dunnett post hoc test corrections, respectively. Analysis of the mean arterial blood pressure assay was performed using Student’s t-test for paired samples. A P value of less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) and SPSS 11.0 statistical package (SPSS Inc., Chicago, IL, USA) softwares. 3. Results 3.1. Kallikrein activation by LOCBE-treated plasma Human plasma treated with LOCBE significantly enhances the hydrolysis of the kallikrein substrate S2302 by plasma in a dose-dependent manner (Fig. 1). The low activity of plasma or LOCBE alone upon S2302 means that the venom interaction with plasma proteins is necessary to display kallikrein activity. 3.2. Kininogenase activity of LOCBE Besides kallikrein activation, LOCBE displays a direct kininogenase activity upon LMWK. Addition
ARTICLE IN PRESS 666
C.B. Bohrer et al. / Toxicon 49 (2007) 663–669
paw induces an edema that persistes during all the course of the experiment. This dose was chosen because it produces an edematogenic response without hemorrhagic effects (de Castro Bastos et al., 2004). Twenty four hours after LOCBE injection, paw edema disappears (data not shown). Pretreatment of the animals with aprotinin or HOE-140 significantly reduces the paw volume (Fig. 3). The inhibitory effect was partial and persistent for both drugs. On the other hand, intravenous LOCBE injection leads to a hypotensive effect that is completely blocked by aprotinin or HOE 140 (Fig. 4).
4. Discussion Fig. 1. Activation of the plasma contact system by Lonomia obliqua caterpillar bristles extract (LOCBE). Group A, 20 mM HEPES buffer pH 7.4; group B, 50 mL of diluted (1:10) human plasma and 20 mM HEPES buffer pH 7.4; group C, 24 mg of LOCBE and 20 mM HEPES buffer pH 7.4; group D, 12 mg of LOCBE, 50 mL of diluted (1:10) human plasma and 20 mM HEPES buffer pH 7.4; group E, 24 mg of LOCBE, 50 mL of diluted (1:10) human plasma and 20 mM HEPES buffer pH 7.4. The activity upon the synthetic kallikrein substrate H-D-Pro-PheArg-pNA (S-2302) at 0.2 mM (final concentration) was determined using a 96-well microplate reader spectrophotometer at 405nm in a final volume of 100 mL. These results are representative of eight independent experiments. Insert: values (expressed as mean and 7SEM, n ¼ 8) of substrate hydrolysis (pmol/min). Statistical analyses were performed using ANOVA test followed by Dunnett’s post hoc test (*Po0.05, there were statistical differences between all groups).
of LMWK to the bath of guinea pig ileum bioassay did not modify the muscle tone, but when LMWK was incubated with LOCBE, a kinin-like contraction was initiated, similarly to the contraction in response to bradykinin (Fig. 2, panels A and D). The kinin nature of the agonist was confirmed as being Bk, since contractions only occurred in the presence of captopril, which avoid the activity of venom kininases z(Fig. 2, panels A and B). On the other hand, LOCBE alone failed to release kinins from HMWK (Fig. 2, panel C). 3.3. The involvement of the kallikrein– kinin system in the edematogenic and hypotensive responses induced by LOCBE The mechanisms involved in the hypotensive and edematogenic effects of LOCBE were studied. Application of LOCBE (35 mg) to the mouse hind
Like other caterpillars from the Saturnidae family, L. obliqua has phytophagous and gregarious habits. Colonies may be seen inhabiting stems and leafs in forests and rural areas in the state of Rio Grande do Sul, southern Brazil. Human contact with these colonies leads to pain, burning sensation and edema. Patients develop DIC, fibrinolysis and hemorrhagic symptoms, although platelet count usually remains unchanged (Zannin et al., 2003). It has been previously reported that LOCBE displays antithrombotic activity in vivo (Prezoto et al., 2002). The antithrombotic activity seems to be a consequence of a consumption coagulopathy. Indeed, the hemorrhagic syndrome is characterized by a decrease in plasma fibrinogen, plasminogen, prekallikrein and prothrombin (Zannin et al., 2003). In this work, the effects of LOCBE on KKS were studied. It was shown that plasma incubated with LOCBE activates pre-kallikrein. Moreover, LOCBE has kininogenase activity. Incubation of LOCBE with human plasma leads to the activation of plasma pre-kallikrein, showing by the increase of S-2302 hydrolysis (Fig. 1). LOCBE directly releases kinin from LMWK, but not from HMWK (Fig. 2). This difference of specificity of kininogens cleavage is also shown within kallikreins, since HMWK is a better substrate for plasmatic kallikrein (releasing BK) whereas LMWK is a better substrate for tissue kallikrein (releasing kallidin) (Mu¨ller-Esterl et al., 1986). So, LOCBE is able to release kinins from all kininogens, directly from LMWK and indirectly (through plasmatic pre-kallikrein activation) from HMWK. The immediate resulting effect in circulation is a fall in blood pressure and in peripheral tissues is edema and erythema formation. Both
ARTICLE IN PRESS C.B. Bohrer et al. / Toxicon 49 (2007) 663–669
667
Fig. 2. Kininogenase activity of L. obliqua caterpillar bristles extract (LOCBE). The kininogenase activity was measured using guinea pig ileum bioassay as described in Materials and methods section. The responses of muscle contraction was obtained by addition of: (A) 50 mg of LMWK and LOCBE (100 mg) in the presence of captopril (3 mM), (B) 50 mg of LMWK and LOCBE (100 mg) in the absence of captopril, (C) 100 mg of HMWK and LOCBE (100 mg) in the presence of captopril (3 mM) and (D) 50 ng of bradykinin. First arrow indicates the addition of kininogen (panels A–C) or Bk (panel D); the second arrow indicates the addition of LOCBE (panels A–C) to the ileum bath. These results are representative of six independent experiments.
Fig. 3. Mouse paw edema induced by L. obliqua caterpillar bristles extract (LOCBE). Female Swiss mice were injected with 50 mL PBS in left hind paw (control), and in the right hind paw with 35 mg of LOCBE. Thirty minutes before LOCBE or PBS injection mice received: 8 mg/kg of aprotinin, i.p. (triangles); 100 nmol/kg of HOE-140, i.p. (inverted triangles) or PBS (circles). The paw edema was evaluated with a plethysmometer at indicated time intervals. Each point represents the mean of eight animals and vertical lines are the SEM. Statistical analysis was performed using ANOVA test followed by Bonferroni’s post hoc test. (*Po0.001 compared to the control group, PBS.)
effects are derived from kinin B2 receptor activation (Regoli et al., 1993). The participation of KKS in the edema and hypotension due to L. obliqua envenomation was also evaluated in vivo and ex vivo. Intraplantar
LOCBE injection leads to paw edema formation. The edema volume was reduced by 50% in mice previously treated with aprotinin, a plasma kallikrein inhibitor. Similar results were obtained in mice treated with HOE-140, a bradykinin B2 receptor antagonist. Clearly, these results show that contact factors and kinins are involved in the edematogenic action of LOCBE. It has been previously demonstrated that LOCBE edematogenic action is partially mediated by histamine (de Castro Bastos et al., 2004), since loratadine produced a significant inhibition of the paw edema. Thus, L. obliqua venom edematogenic action is also mediate by kinins-releasing, which induces edema. Also, LOCBE is able to induce a hypotensive response mediated by similar mechanisms, because aprotinin and HOE-140 inhibit the fall in the arterial blood pressure elicited by LOCBE. The KKS participation in edema and hypotension was also observed for other venomous animals, like snakes Bothrops lanceolatus (Faria et al., 2001) and Trimeresurus mucrosquamatus (Wang and Teng, 1988), wasp Vespula vulgaris (Griesbacher et al., 1998) and spider Phoneutria nigriventer (Antunes et al., 1993). Despite kinin participation is well characterized for other venoms, it was never shown before for L. obliqua. In addition to the kinin role in the inflammatory responses to L. obliqua envenomation, it may also
ARTICLE IN PRESS 668
C.B. Bohrer et al. / Toxicon 49 (2007) 663–669
envenomation could be related to pro-inflammatory genes up regulated after KKS activation, since activation of B2 receptors is known to activate NFkB and interleukin 1b, which can enhance expression of B1 receptor (Pan et al., 1996). Indeed, bradykinin is known to sensitize nociceptors following the release of cytokines and prostaglandins (Dray and Perkins, 1997). In summary, the results clearly demonstrate that KKS plays a role in the edematogenic and hypotensive effects during L. obliqua envenomation. Undoubtedly, these data show that the contact phase enzymes of the coagulation cascade, mainly plasma kallikrein, plays a role for in the L. obliqua venom toxic effects. Acknowledgments
Fig. 4. Effects of aprotinin and HOE-140 on vasodilatory response to bradykinin or L. obliqua cartepillar bristles extract (LOCBE). After surgery, vessel cannulation, and equilibration period, a response to bradykinin (3 nmol/kg, i.v., left) or LOCBE (3 mg/kg, i.v., right) were evaluated before (open bar) and 10 min after (filled bar) the intravenous bolus injection of aprotinin (4 mg/kg, panel A) or HOE-140 (100 nmol/kg, panel B). Each point represents the mean 7SEM of six animals. Statistical comparison was performed in relation to the corresponding effect obtained before HOE-140 or aprotinin injection. Statistical analysis was performed using Student’s t-test for paired samples (*Po0.05).
be involved in hyperalgesic response, since Bk is a powerful pain mediator (Wang et al., 2006). De Castro Bastos et al. (2004) described that LOCBE nociceptive effects are mediated mainly by prostaglandin release, since it was strongly inhibited by indomethacin pretreatment. These authors proposed that the phospholipase A2 activity present in LOCBE (Arocha-Pin˜ango et al., 2000; Seibert et al., 2003) could be responsible for the prostaglandin release. The observation of a delayed type nociceptive response after LOCBE administration (de Castro Bastos et al., 2004) corroborates a possible functional role of kinins in the nociceptive action of LOCBE. The nociceptive aftersensation caused by
We thanks to Conselho Nacional de Pesquisa (CNPq), Fundac- a˜o de Amparo a` Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Coordenac- a˜o de Aperfeic- oamento de Pessoal de Nı´ vel Superior (CAPES), Programa Nacional de Cooperac- a˜o Acadeˆmica (PROCAD) and Programa Nacional de Exceleˆncia (PRONEX) for financial support. Special thanks to Prof. Joa˜o Feliz Duarte de Moraes (Departamento de Estatı´ stica—Universidade Federal do Rio Grande do Sul, Brazil).
References Antunes, E., Marangoni, R.A., Giglio, J.R., Brain, S.D., de Nucci, G., 1993. Activation of tissue kallikrein–kininogen–kinin system in rabbit skin by a fraction isolated from Phoneutria nigriventer (armed spider) venom. Toxicon 31 (11), 1385–1391. Arocha-Pin˜ango, C.L., Marval, E., Guerrero, B., 2000. Lonomia obliqua genus caterpillar toxins: biochemical aspects. Biochimie 82, 937–942. Arocha-Pin˜ango, C.L., Guerrero, B., 2001. Lonomia genus caterpillar envenomation: clinical and biological aspects. Haemostasis 31, 288–293. Brown, R.E., 1989. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem. 180, 136–139. de Castro Bastos, L., Veiga, A.B.G., Guimara˜es, J.A., Tonussi, C.R., 2004. Nociceptive and edematogenic responses elicited by a crude bristle extract of Lonomia obliqua caterpillars. Toxicon 43 (3), 273–278. Coˆrrea, M.S., Siqueira-Batista, R., Gomes, A.P., FrancoBarbosa, A., Verzola, A.C.A., Oliveira, F.R.Q., Squeff, F.A., Motta-Leal-Filho, J.M., Tavares, R.H., Amorim, D.S., De-Maria-Moreira, N.L., Santos, S.S., 2004. Lonomia erucism in Tereso´polis, Rio de Janeiro State, Brazil. Report of
ARTICLE IN PRESS C.B. Bohrer et al. / Toxicon 49 (2007) 663–669 a probable case and review. Rev. Soc. Bras. Med. Trop. 37 (5), 418–421. Donato, J.L., Moreno, R.A., Hyslop, S., Duarte, A., Antunes, E., Le Bonniec, B.F., Rendu, F., de Nucci, G., 1998. Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin. Thromb. Haemost. 79, 539–542. Dray, A., Perkins, M.N., 1997. Kinins in pain. In: Farmer, S. (Ed.), The Kinin System. Academic Press, San Diego, pp. 157–172. Ferreira, S.H., 1979. A new method for measuring variation of rat paw volume. J. Pharm. Pharmacol. 31 (9), 648. Faria, L., Antunes, E., Bon, C., Araujo, A.L., 2001. Pharmacological characterization of the rat paw edema induced by Bothrops lanceolatus (Fer de lance) venom. Toxicon 39 (6), 825–830. Griesbacher, T., Althuber, P., Zenz, M., Rainer, I., Griengl, S., Lembeck, F., 1998. Vespula vulgaris venom: role of kinins and release of 5-hydroxytryptamine from skin mast cells. Eur. J. Pharmacol. 351 (1), 95–104. Herald, H., Morgelin, M., Bjorck, L., 2003. Contact activation by pathogenic bacteria: a virulence mechanism contributing to the pathophysiology of sepsis. Scand. J. Infect. Dis. 35. (9), 604–607. Kaplan, A.P., Silverberg, M., 1987. The coagulation–kinin pathway in human plasma. Blood 70, 1–16. Mu¨ller-Esterl, W., Iwanaga, S., Nakanishi, S., 1986. Kininogens revisited. Trends Biochem. Sci. 11 (8), 336–339. Pan, Z.K., Zuraw, B.L., Lung, C.C., Prossnitz, E.R., Browning, D.D., Ye, R.D., 1996. Bradykinin stimulates NF-kB activation and interleukin 1b gene expression in cultured human fibroblasts. J. Clin. Invest. 98, 2042–2049. Pierce, J.V., Guimara˜es, J.A., 1974. Further characterization of highly purified human plasma kininogens. In: Pisano, J.J., Austen, K.F. (Eds.), Chemistry and Biology of the Kallikrein–kinin System in Health and Disease. DHEW Publication, Maryland, pp. 121–127. Pinto, A.F.M., Dobrovolski, R., Veiga, A.B.G., Guimara˜es, J.A., 2004. Lonofibrase, a novel alpha-fibrinogenase from Lonomia obliqua caterpillars. Thromb. Res. 113 (2), 147–154.
669
Pinto, A.F.M., Silva, K.R.L.M., Guimara˜es, J.A., 2006. Proteases from Lonomia obliqua venomous secretions: comparison of procoagulant, fibrin(ogen)olytic and amidolytic activities. Toxicon 47 (1), 113–121. Prezoto, B.C., Maffei, F.H.A., Mattar, L., Chudzinski-Tavassi, A.M., Curi, P.R., 2002. Antithrombotic effect of Lonomia obliqua caterpillar bristle extract on experimental venous thrombosis. Braz. J. Med. Biol. Res. 35 (6), 703–712. Regoli, D., Jukic, D., Gobeil, F., Rhaleb, N.E., 1993. Receptors for bradykinin and related kinins: a critical analysis. Can. J. Physiol. Pharmacol. 71 (8), 556–567. Seibert, C.S., Shinohara, E.M., Sano-Martins, I.S., 2003. In vitro hemolytic activity in Lonomia obliqua caterpillar bristles extract on human and Wistar rat erythrocytes. Toxicon 41, 831–839. Veiga, A.B.G., Blochtein, B., Guimara˜es, J.A., 2001. Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidoptera, Saturniidae). Toxicon 39, 1343–1351. Veiga, A.B.G., Pinto, A.F.M., Guimara˜es, J.A., 2003. Fibrinogenolytic and procoagulant activities in the hemorrhagic syndorme caused by Lonomia obliqua caterpillars. Thromb. Res. 111 (1-2), 95–101. Wang, J.P., Teng, C.M., 1988. Rat paw edema induced by trimucase II, a kinin-releasing enzyme from Trimeresurus mucrosquamatus venom. Eur. J. Pharmacol. 157 (1), 61–66. Wang, H., Ehnert, C., Brenner, G.J., Woolf, C.J., 2006. Bradykinin and peripheral sensitization. Biol. Chem. 387 (1), 11–14. Webster, M.E., Prado, E.S., 1970. Glandular kallikrein from horse and human urine and from hog paˆncreas. Methods Enzymol. 19, 681–699. Zannin, M., Lourenc- o, D.M., Motta, G., Dalla Costa, L.R., Gamborgi, G.P., Noguti, M.A., Chudzinski-Tavassi, A.M., 2003. Blood coagulation and fibrinolytic factors in 105 patients with hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillar in Santa Catarina, southern Brazil. Thromb. Haemost. 89 (2), 355–364.
