Losac, a factor X activator from Lonomia obliqua bristle extract: Its role in the pathophysiological mechanisms and cell survival

BBRC Biochemical and Biophysical Research Communications 343 (2006) 1216–1223 www.elsevier.com/locate/ybbrc

Losac, a factor X activator from Lonomia obliqua bristle extract: Its role in the pathophysiological mechanisms and cell survival Miryam Paola Alvarez Flores, Ma´rcio Fritzen, Cleyson V. Reis, Ana Marisa Chudzinski-Tavassi * Laborato´rio de Bioquı´mica e Biofı´sica, Instituto Butantan, Sa˜o Paulo, Brazil Received 26 February 2006 Available online 20 March 2006

Abstract Contact with the bristles of the caterpillar Lonomia obliqua can cause serious hemorrhage. Previously it was reported that a procoagulant protein (Lopap) in the bristle extract of L. obliqua increases cell longevity by inhibiting apoptosis. In this work, we purified from bristle extract a factor X activator that stimulates proliferation of endothelial cells. This protein, named Losac, was purified by ion exchange chromatography, followed by gel filtration chromatography and reverse-phase HPLC. Losac is a 45-kDa protein that activates factor X in a concentration-dependent manner and does not depend on calcium ions. In cultures of HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. HUVECs incubated with Losac (0.58 lM for 1 h) increased release of nitric oxide and tissue-plasminogen activator, which both may mediate anti-apoptosis. Losac also increased slightly the decay-accelerating factor (DAF = CD55), which protects cells from complement-mediated lysis. On the other hand, Losac did not alter the release or expression of von Willebrand factor, tissue factor, intercellular adhesion molecule-1, interleukin-8, and prostacyclin. These characteristics indicate that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. Losac may have biotechnological applications, including the reduction of cell death and consequently increased productivity of animal cell cultures, and the use of hemolymph of L. obliqua for this purpose is already being explored. Further study is required to elucidate the mechanism for the inhibition of apoptosis by Losac.  2006 Elsevier Inc. All rights reserved. Keywords: Lonomia obliqua; Losac; Coagulation; Endothelial cell; HUVEC; Factor X activator

Contact with the bristles of Lonomia obliqua, a caterpillar of the family Saturniidae that is found in the South of Brazil, can cause a hemorrhagic syndrome [1]. Zannin et al. [2] reported that patients who had contact with this caterpillar presented a severe depletion of coagulation factors, as well as a secondary fibrinolysis. Extracts of the bristles contain procoagulant proteins such as activators of prothrombin and of factor X [3,4]. We have previously isolated from bristle extract a prothrombin activator named Lopap that induces consumptive coagulopathy in rats and may contribute to the hemorrhagic syndrome that *

Corresponding author. Fax: +55 11 3726 1024. E-mail address: [email protected] (A.M. ChudzinskiTavassi). 0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.068

is observed in human patients [4–6]. Recently a L. obliqua protein with FXa-like activity [7] with the same sequence as Lopap [5] was described. The factor responsible for the FX activating activity of L. obliqua bristles has not been isolated so far. FX activators are found in viper venoms, but only a limited number of these have been isolated. A well-known example is RVVX, a metalloprotease from Vipera russelli [8], which, like other FX activators from snakes, depends on Ca2+ [9]. Previously it was shown that hemolymph from some insects can increase cell longevity by inhibiting apoptosis in insect cell cultures [10]. It was shown that the hemolymph of L. obliqua contains anti-apoptotic and growthpromoting factors [11,12]. Recently it was reported that Lopap, a prothombin activator purified from bristles

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extract, increases longevity of human endothelial cells by inhibiting apoptosis [13,14]. In this study, we purified the FX activator from the bristles of L. obliqua. We examined the effect of the purified protein on proliferation and apoptosis in cultured cells. In addition, we measured the effect of the purified protein on the expression and release of a range of proteins involved in fibrinolysis, thrombosis, inflammation, and blood coagulation. Materials and methods Preparation of bristle extract. Lonomia obliqua caterpillars were collected in the south of Brazil (states of Santa Catarina, Rio Grande do Sul, and Parana´) and frozen in dry ice. The bristles were harvested by cutting them at their base. The bristles were ground in a mortar, homogenized in PBS, pH 7.4, and the insoluble material was removed by centrifugation for 10 min at 2000g at 4 C. Purification of the factor X activator. The bristle extract was applied to a DEAE-Sephadex A-50 column (2.8 · 6 cm, Amersham Bioscience, Uppsala, Sweden). The extract was eluted with a non-linear gradient from 0.02 to 0.5 M NaCl in 20 mM Tris–HCl at pH 8.0 at a flow rate of 120 ml/ h, and fractions of 2 ml were collected. Fractions that activated FX were dialyzed against 50 mM Tris–HCl containing 100 mM NaCl at pH 8.0. The dialyzed pool was submitted to gel filtration chromatography (TSKGel 3000 SW column, 7.5 mm · 60 cm, TosoHaa Germany, MerckHitachi D-2500 HPLC system, and Shimadzu SPD-6AV UV monitor with detection at 214 nm). The sample was eluted with 50 mM Tris–HCl containing 100 mM NaCl at pH 8.0 at a flow rate of 36 ml/h. Fractions that activated FX were pooled and loaded again on the same filtration column and in the same buffer at a flow rate of 24 ml/h. The fractions with FX activating activity were subjected to reversed-phase HPLC with a C4 column (4.6 · 250 mm, J.T. Baker, Phillipsburg, NJ, USA). Elution solvents were 0.1% TFA in water (solvent A) and acetonitrile with 10% solvent A (solvent B). A linear gradient of solvent B (0–70%) was applied at a flow rate of 1.0 ml/min for 30 min. The optical density of the fractions was measured at 214 nm. The purified FX activator eluted from RP-HPLC was analyzed by 12% (w/v) SDS–PAGE stained with Coomassie brilliant blue R250. Protein concentration was determined by measuring the absorbance at 214 nm or with a protein assay kit from Bio-Rad Laboratories. Bovine serum albumin was used as standard. Measurement of factor X activation. The purified protein (29–107 nM) was incubated with 0.2 lM human FX (Sigma Chemical, St. Louis, MO, USA) and 25 mM Tris–HCl, pH 8.3, for 20 min at 37 C in a total volume of 195 ll. Then 5 ll of 8 mM S-2765 (Chromogenix, Mo¨lndal, Sweden) was added. The amount of p-nitroaniline produced by FXa during 20 min at 37 C was measured at 405 nm in a Spectra MAX 190 microplate reader (Molecular Devices, USA). Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) were obtained by digestion of umbilical cord veins with collagenase (Worthington Biochemical, USA) as described previously [15]. Cells were seeded into culture flasks (25 cm2) precoated with 2% (w/v) gelatin (ICN Biomedicals, Costa Mesa, CA, USA). Cells were grown in RPMI 1640 medium (Cultilab, Campinas, Brazil) containing 10% fetal bovine serum (FBS, Cultilab, Brazil), 100 IU penicillin/ 100 lg/ml streptomycin solution (Cultilab, Brazil), 2 mM L-glutamine, 45 lg/ml heparin, 1 mM sodium pyruvate, 50 lM 2-mercaptoethanol, and 25 lg/ml endothelial cell growth supplement from bovine neural tissue (ECGF) (Sigma, USA) at 37 C in a humidified incubator with 5% CO2. Endothelial cells between first and third passage were used for all experiments. Cells to be used for the measurement of expression of surface receptors and release of chemical mediators were plated on 24-well dishes, coated with 2% gelatine, and grown to confluence. Confluent cells were detached with 0.25% trypsin/0.02% EDTA solution (Cultilab, Brazil), washed with 10% FBS-supplemented PBS (Ca2+ and Mg2+ free) (PBS/FBS), and

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centrifuged for 10 min at 400g at 4 C. To exclude lipopolysaccharide interference, some experiments were performed in the presence of 7 lg/ml polymyxin B (Sigma, USA). Cell proliferation assays. HUVECs (4 · 103 cells/well) were plated in gelatin-coated 96-well flat-bottomed tissue cluster plates in RPMI 1640 medium supplemented with 10% FBS and allowed to attach overnight. Medium was then replaced by fresh medium (RPMI 1640 with 10% FBS, 100 IU penicillin, and 100 lg/ml streptomycin) in the absence or presence of the purified protein (0.12 or 0.58 lM). After 72 h in culture, cells were detached from the wells with trypsin/EDTA solution, stained with trypan blue, and counted in a Neubauer chamber under a phase-contrast microscope. Detection of apoptosis. Apoptosis was induced by serum deprivation. Cells were kept in RPMI containing 1% FBS for 48 h in the absence or in the presence of the purified protein (0.12 or 0.58 lM). Apoptosis was evaluated by two methods. First, by flow cytometry using histogram analysis to identify hypodiploid nuclei. Adherent and floating cells were pooled, washed, and fixed in cold 70% ethanol. After centrifugation, cellular DNA was stained with 100 lg/ml propidium iodide (Calbiochem, La Jolla, CA, USA) in 0.1% Triton X-100, 1 mM EDTA. After addition of 200 lg/ml DNase-free RNase (Sigma, USA), cells were analyzed in a FACScan cytofluorometer (Becton–Dickinson, Mountain View, CA, USA). Second, nuclear endothelial cell changes and necrosis were evaluated by fluorescent microscopy using 100 lg/ml acridine orange (Aldrich Chemical, St. Louis, MO, USA) and 100 lg/ml ethidium bromide (Sigma, USA). Cells with fragmented nuclei were defined as apoptotic. Nitric oxide assay. Total nitric oxide (NO) production was measured by the accumulation of nitrites and nitrates into the HAM F12 (Cultilab, Brazil) culture medium. HUVECs were incubated for 1 h with 0.12 or 0.58 lM of the purified protein. The supernatants were centrifuged for 10 min at 400g at 4 C and aliquots of 10 ll were injected into the NO analyzer (NOA280; Sievers Instrument, Boulder, CO, USA). In the NO analyzer, nitrites and nitrates in the supernatants were reduced to NO with 8 mg/ml vanadium chloride (Sigma, USA) in 1 M HCl at 90 C, and the NO formed was detected by gaseous phase chemiluminescence after reaction with ozone. NO concentration was calculated from a sodium nitrate standard curve. Measurement of ICAM-1 and DAF expression. HUVECs were incubated for 1 h with 0.12 or 1.12 lM of the purified protein or 1 ng/ml TNF-a (BD Biosciences Pharmigen, San Diego, CA, USA). The medium was removed and the cells were cultured in serum-free RPMI medium for another 12 h for expression of intercellular adhesion molecule-1 (ICAM1 = CD54) or 24 h for expression of decay accelerating factor, DAF = CD55. Then cells were treated with trypsin/EDTA solution and centrifuged at 400g for 10 min at 4 C. The pellets were resuspended in 50 ll PBS/FBS (10%) containing saturating concentrations of phyicoerythrin-conjugated anti-CD54, anti-CD55 or equivalent concentrations of an isotypic control IgG1 (BD Biosciences, USA). After incubation for 30 min at 4 C, cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry in a FACScan cytofluorometer (Becton–Dickinson, Mountain View, CA, USA). Appropriate settings of forward and side scatter gates were used to examine 5000 cells per experiment. Measurement of PGI2, IL-8, vWF, and t-PA. After treatment of HUVECs with 0.12 or 0.58 lM of the purified protein or 5 U/ml thrombin (Sigma, USA) for 1 h, medium was removed and the cells were cultured for 24 h in RPMI containing 10% FBS. Supernatants removed after 1 and 24 h were centrifuged for 10 min, 400g at 4 C to remove cellular debris. The content of interleukin-8 (IL-8) and tissue-plasminogen activator (t-PA) in the supernatant was measured with ELISA kits from Oncogene Reseach Products (San Diego, CA, USA). Von Willebrand Factor (vWF) was measured using an ELISA kit from Diagnostica Stago (Asnieres, FR). Prostacyclin (PGI2) production was measured by the measurement of the stable PGI2 metabolite 6-keto-prostaglandin F1a with an ELISA kit from Cayman Chemical (Ann Arbor, MI, USA). Measurement of tissue factor activity. Tissue factor activity (TF) was assayed by measuring the enzymatic activation of factor X by the TF/factor VIIa complex on confluent HUVEC. Adherent cells were incubated for 4 h

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at 37 C in RPMI 1640 medium in the presence of the purified protein (0.12 or 0.58 lM) or 10 ng/ml TNF-a. The cells were washed with Hepes-buffered saline (50 mM Hepes, 150 mM NaCl, and 5 mM CaCl2, pH 7.5) and incubated for 30 min at room temperature in the presence of 5 nM FVIIa (Calbiochem, USA) and 400 nM FX in Hepes-buffered saline. The reaction was stopped by adding EDTA (10 mM). The activated FX produced by the TF/factor VIIa complex was measured after incubation for 1 h at room temperature with 200 lM of S2222 (Chromogenix, Sweden), a chromogenic substrate specific to FXa. The absorbance of the samples was measured at 405 nm in a Spectra MAX 190 microplate reader. The data were expressed as a percentage of the value of untreated HUVEC. Statistical analysis. All results are expressed as means ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Newman–Keuls multiple comparison procedure. Values of p lower than 0.05 were considered statistically significant.

Results Isolation of a factor X activator from bristle extract of L. obliqua The FX activator was purified by a three-step procedure. The chromatogram of the bristle extract (20.5 mg, specific activity 12 U/mg) after ion exchange chromatography is shown in Fig. 1A. Fractions 8–93 (peak I) presented FX activating activity. This pool accounted for 10% of the dry weight (2 mg, specific activity 46.9 U/mg) of the

original sample. In addition, fractions 733–771 (peak V) presented prothrombin activation activity. Fractions with FX activation activity (peak I) were collected and subjected to gel filtration chromatography. The chromatogram contained 9 peaks (Fig. 1B). The third peak, which was collected between 23.05 and 29.80 min, presented FX activation activity (204 lg, specific activity of 147 U/mg). The active fractions were pooled and submitted to a second gel filtration step on the same column, resulting in six peaks (Fig. 1C). Only peak III (69 lg), which was collected between 36.02 and 40.57 min, presented FX activation activity. The specific activity of this peak was 203 U/mg, about 17 times higher than that of the crude venom. We submitted this peak to reversed-phase HPLC to eliminate contaminants. The result was a single sharp peak (Fig. 2A) with FX activator activity. Analysis of the purified protein by SDS–PAGE in 12% gels under non-reducing conditions resulted in a band with a molecular weight of 45 kDa (Fig. 2B). This band was responsible for the activation of FX. The FX activator was named Losac (Lonomia obliqua Stuart-factor Activator). Fig. 3 shows that Losac induced a dose-dependent activation of FX, as measured from amidolytic activity of activated FX. However, even the highest dose of Losac used

Fig. 1. Purification of the factor X activator from extract of bristles of the Lonomia obliqua caterpillar. (A) Ion exchange chromatogram with a DEAESephadex A-50 column. (B) First gel filtration chromatogram with a TSK-Gel 3000 SW column. (C) Second gel filtration chromatogram on the same column.

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Fig. 2. Reverse-phase-HPLC analysis of the factor X activator. (A) Reverse-phase chromatogram with a C4 column (HPLC system) of peak III in Fig. 1C. (B) SDS–PAGE under non-reducing conditions. Lane 1: 20 lg Losac from RT-HPLC. Lane 2: molecular size standard containing phosphorylase B (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21.5 kDa), and a-lactoalbumin (14.4 kDa). Lane 3: 100 lg crude bristle extract.

Fig. 3. Effects of Losac concentration on activation rate of factor X. Factor X activation was measured as described under Materials and methods. Factor Xa formation was followed spectrophotometrically by recording the liberation of p-nitroaniline at 405 nm during 20 min at 37 C. Losac: () 29 nM. (·) 58 nM. (j) 80 nM. (d) 107 nM. (m) Losac (107 nM) without FX (negative control).

(107 nM) did not cause maximal activation of FX. Unlike FX activators from snake venom [9], Losac did not require calcium ions for the activation of FX. Losac induces endothelial cell proliferation and inhibits apoptosis The previously purified procoagulant protein from L. obliqua bristles, Lopap, activates the coagulation system and triggers endothelial cell responses [13,14]. Therefore, we studied the effect of Losac on cell viability and apoptosis. Incubation of quiescent endothelial cells (cultured in RPMI containing 10% FBS) for 72 h with Losac caused a dose-dependent increase in the number of cells (Fig. 4). Losac also minimized cell death caused by nutrient depletion. Fluorescence microscopy showed that HUVEC kept in serum-deprived conditions for 48 h (RPMI containing 1% FBS) had fewer apoptotic cells with Losac

Fig. 4. Effect of Losac on endothelial cell proliferation. Number of endothelial cells after 72 h of incubation in RPMI containing 10% FBS with two different concentration of Losac (n = 5, *p < 0.05 vs. control).

in the culture medium [55.5 ± 4.3, 33.7 ± 2.1*, and 29.2 ± 5.7*% of apoptotic cells, for control, and 0.12 and 0.35 lM Losac, respectively, n = 6, *p < 0.05 vs. control]. Cultures kept for 48 h in quiescent conditions also contained fewer apoptotic cells when Losac was present [20.3 ± 1.37, 18.7 ± 1.6, and 12.4 ± 2.2*% of apoptotic cells, for control, and 0.12 and 0.35 lM Losac, respectively, n = 6, *p < 0.05 vs. control]. The DNA content of serum-deprived HUVEC after 48 h of incubation in the absence or presence of Losac was analyzed by flow cytometry (Fig. 5). In quiescent conditions, most cells were in the G1 phase (Fig. 5A). In cells deprived of nutrients, the number of cells in the G1 phase was reduced, and a population of hypodiploid (apoptotic) cells was present (Fig. 5B). In contrast, in nutrient-depleted cells incubated with 0.12 lM Losac, the amount of apoptotic DNA was reduced [48.1 ± 3.1 vs. 28.3 ± 1.3* % of apoptotic cells for control and Losac, respectively, n = 3, *p < 0.05 vs. control], and the number of cells in the S phase was increased (Fig. 5C). Cell necrosis in control or proteasetreated-HUVEC was never higher than 2% (n = 6).

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Fig. 6. Losac stimulates NO production. HUVECs cells were incubated for 1 h in HAM F12 medium with 0, 0.12 or 0.58 lM Losac or with thrombin. NO was determined in culture supernatants. Bars represent the mean ± SEM of six independent experiments. *p < 0.05 vs. control.

leukocytes into tissues [20]. As shown in Table 1, Losac did not increase the expression of ICAM-1 on the cell membrane. However, Losac slightly increased the decayaccelerating factor (DAF = CD55), which protects cells from complement-mediated lysis [21]. Tumor necrosis factor-a (TNF-a) stimulated the expression of these molecules, as expected. Fig. 5. Effect of Losac on the cell cycle of serum-deprived HUVEC. (A) HUVECs kept for 48 h in serum-deficient RPMI medium (1% FBS) increased the number of apoptotic cells, as determined by flow cytometry on cells stained with propidium iodide. (B) HUVECs in RPMI containing 10% FBS were mostly in the G1 phase. (C) Apoptosis induced by serum deprivation was prevented by 0.12 lM Losac.

Losac triggers NO liberation but not PGI2 release NO and PGI2 are potent vasodilators, inhibitors of platelet activation, and inhibitors of smooth muscle cell proliferation [16]. NO may also induce angiogenesis in endothelial cells by inhibiting apoptosis [17–19]. For these reasons we examined the liberation of both molecules from HUVEC. HUVEC incubated for one hour with Losac presented a significant increase of nitric oxide production (Fig. 6 and Table 1). However, Losac did not affect the release of PGI2 (Table 1), whereas incubation of HUVEC for 1 h with 5 U/ml of thrombin increased PGI2 release. Subsequent incubation of HUVEC pre-treated with Losac in Losac-free medium for 24 h did not significantly increase PGI2 concentration [3060 ± 396.3 vs. 3235 ± 270 pg/ml for control and 0.58 lM Losac, respectively, n = 3]. HUVEC increased PGI2 after pre-incubation for 1 h with 5 U/ml thrombin [3060 ± 396.3 vs. 6249 ± 396 pg/ml, n = 3]. ICAM-1 and DAF expression ICAM-1 is a cell adhesion molecule that aids recruitment, adhesion, and trans-endothelial migration of

Losac stimulates secretion of pre-formed t-PA but not of vWF and IL-8 We next analyzed the effect of Losac on the release of molecules involved in fibrinolysis (t-PA), thrombosis (vWF), and inflammation (IL-8). These molecules are known to be stored in Weibel-Palade bodies [22]. One hour of incubation with Losac increased t-PA secretion in a dose-dependent manner (p < 0.05, Fig. 7 and Table 1). Endothelial cells pretreated with Losac for 1 h and cultured for 24 h in Losac-free medium did not increase t-PA release compared to untreated cells [2284 ± 85 vs. 2365 ± 96 pg/ml for control and 0.58 lM Losac, respectively, n = 3]. This suggests that Losac stimulated the release of t-PA from pre-formed stores, but not the synthesis of new t-PA. The positive control thrombin, a well-characterized agonist for t-PA release from Weibel-Palade bodies [23], increased, as expected, new synthesis of t-PA [2284 ± 85 vs. 3239 ± 259* pg/ml for control and 5 U/ml thrombin, respectively, n = 3, *p < 0.05]. Although Losac tended to increase the release of von Willebrand Factor (vWF), which is the major component inside Weibel-Palade bodies [24], the effect was not statistically significant (Table 1). The positive control, thrombin (5 U/ml), almost doubled vWF release. Losac also did not seem to stimulate vWF synthesis, since cells incubated for 1 h with Losac and cultured in Losac-free medium for the subsequent 24 h did not produce increased levels of vWF (data not shown).

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Table 1 Effect of Losac on the production of molecules involved in coagulation and endothelial cell functions Molecule

n

Control

Losac 0.12 lM

Losac 0.58 lM

Losac 1.12 lM

Thrombin 5 U/ml

TNF-a 1 ng/ml

NO (lM) PGI2 (pg/ml) ICAM-1 (AUF) DAF (AUF) t-PA (pg/ml) vWF (ng/ml) IL-8 (pg/ml) TF (%)a

6 3 5 5 3 3 3 3

6.5 ± 0.6 203 ± 39 13.6 ± 2.2 53 ± 3 72 ± 4 15.4 ± 2.2 204 ± 13 100 ± 13

10.3 ± 1.7 160 ± 33 14.6 ± 2.2 49.5 ± 6.2 130 ± 7 16.1 ± 4.5 181 ± 12 122 ± 22

23.8 ± 2.7 234 ± 43 NT NT 184 ± 18* 22.1 ± 3.0 202 ± 16* 120 ± 18

NT NT 16.3 ± 5.3 78.4 ± 12 NT NT NT NT

18.4 ± 1.2 3746 ± 568* NT NT 233 ± 12.5* 32 ± 6* 358 ± 28* NT

NT NT 54.4 ± 3.8* 93.4 ± 6* NT NT NT 186 ± 15*

All molecules were quantified in supernatants after 1 h of incubation of HUVEC with Losac. NT, not tested; AUF, arbitrary units corresponding to the mean intensity of fluorescence of molecules per cell. a For TF assays the TNF-a concentration was 10 ng/ml. * p < 0.05.

Fig. 7. Losac induces t-PA release from cultured HUVECs. Confluent cells in RPMI containing 10% FBS were incubated for 1 h in the absence or presence of 0.12 or 0.58 lM Losac or thrombin. t-PA concentration in the supernatants was measured. Bars represent the mean ± SEM of three independent experiments. *p < 0.05 vs. control.

Losac did not affect the release of IL-8 (Table 1), whereas 5 U/ml of thrombin increased release of IL-8. IL-8 synthesis was also not stimulated (data not shown). Losac did not increase TF activity in HUVEC TF expression and activity can be induced in endothelial cells by various stimuli including cytokines, growth factors, and biogenic amines [25]. As shown in Table 1, Losac failed to modify the TF pro-coagulant activity on the HUVEC surface. As expected, the positive control TNF-a, a proinflammatory cytokine, enhanced TF activity by 86% (*p < 0.05). Discussion Hemorrhagic syndrome seems to be the most important clinical complication produced in people who experienced contact with bristles of L. obliqua [1]. Recently it was reported that the consumptive coagulopathy in patients envenomed by contacting this caterpillar is a consequence of severe depletion of coagulation factors, as well as secondary activation of fibrinolysis [2]. In vitro studies showed that crude bristle extract from L. obliqua induces

clot formation by triggering activation of both prothrombin and Factor X [1,3]. Recently it was reported that the extract of bristles from L. obliqua contains a prothrombin activator, a Ca2+-independent activator of FX, and a FXalike protein [3,5,7]. In this study, we purified a 45-kDa protein that activated FX in the absence of calcium ions. This protein differs from the FX activators from snake venom, which critically depend on the presence of calcium ions [9]. Losac apparently acts mostly on FX, since it did not hydrolyze chromogenic substrates to FXa (S-2765, S-2222), thrombin (S-2238) or plasmin (S-2251) (data not shown). Losac also did not act on prothrombin (data not shown). Activators of FX have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species [9]. Losac is the first FX activator purified from a lepidopteran secretion. Losac is not the only procoagulant protein isolated from L. obliqua bristles. We recently described Lopap, a protein that not only activates the coagulation system [4–6] but also stimulates the liberation of molecules involved in coagulation and inflammatory mechanism from endothelial cells [13], and improves cell survival and reduces cell death probably by stimulating the release of molecules such as NO [14]. Among other functions, vascular endothelial cells synthesize potent procoagulant and anti-thrombotic proteins, which can directly initiate receptor-coupled cellular signalling and cell activation [16]. Several procoagulant proteins isolated from snake venoms have been reported to affect endothelial cell function [26]. Therefore, we investigated the effect of Losac on endothelial cell function. Our results have shown that, beyond its role in coagulation as a procoagulant protein, Losac dose-dependently induced the proliferation of HUVEC. Moreover, in nutrient-depleted cells exposed to Losac, the amount of apoptotic DNA was reduced and the number of cells in the S phase was increased, as determined by fluorescence microscopy and flow cytometry. These results suggest that Losac can stimulate DNA duplication even in death conditions.

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In an attempt to find other activities of Losac on endothelial cells, we examined its effect on the release of molecules involved in fibrinolysis, thrombosis, blood coagulation, and inflammation. t-PA was released by Losac in a dose-dependent manner. However, synthesis of new t-PA was not stimulated. This serine protease cleaves plasminogen into its biologically active form, plasmin [27]. Previous studies have suggested that the ability of t-PA to activate plasminogen is important during angiogenesis [28]. However, t-PA can also increase endothelial cell proliferation independent of plasmin generation and therefore may be mediated by a t-PA receptor [29]. It is possible that the liberation of pre-formed stores of t-PA may contribute to the proliferative effect of Losac. It is well established that the inflammatory and coagulation pathways are invariably linked. A large number of cytokines such as TNF-a, and mediators such as thrombin, interact with specific receptors and elicit inflammatory responses, including leukocyte migration and chemotaxis, and induce the expression of prothrombotic molecules [16,22,25]. In our study, Losac did not up-regulate IL-8 or ICAM-1. IL-8 is a leukocyte-specific chemoattractant [30] and ICAM-1 is a cell adhesion molecule that mediates adhesion of leukocytes to activated endothelium [20]. Thus, Losac probably does not affect the inflammatory mechanism on endothelial cells. Moreover, our results also indicate that Losac has no effect on the expression of prothrombotic molecules such as TF and vWF. TF is a single-chain protein expressed at surface of endothelial cells, initiates blood coagulation by forming complex with factor VIIa which activates substrate factors IX, X, and VII by proteolytic activation [25,31]. vWF, a multimeric plasma glycoprotein, is required for platelet binding to the subendothelium during hemostasis, serves as a carrier for coagulation factor VIII, and is involved in endothelial cell adhesion to the basal lamina of the vessel [22]. It is surprising that Losac increased the release of t-PA without stimulating vWF and IL-8 because all these proteins are thought to be stored together in Weibel-Palade bodies [24,30,32]. The nature of such a selective release remains to be determined. However, it was suggested that t-PA and vWF may be stored in different secretory granules, since the mechanisms involved in the secretion of these two proteins differ in some aspects [33]. NO and PGI2 are potent vasodilators and inhibitors of platelet activation [16]. In our studies Losac modulated NO liberation, but not PGI2 production. The ability of Losac to stimulate NO release, but not PGI2, may contribute to its anti-apoptotic effects, since NO inhibits apoptosis and stimulates endothelial cell proliferation [17–19]. DAF expression was slightly, but not significantly, enhanced with high concentration of Losac. DAF is a surface receptor that protects cells from complement-mediated lysis by accelerating the decay of the classical and alternative pathway C3 and C5 convertases [21]. It is possible that

up-regulation of DAF could represent a mechanism of endothelial protection. It has been reported that the hemolymph of L. obliqua has anti-apoptotic and cell survival properties, and may have biotechnological applications in cell culture [11,12]. Recently Pinto et al. [34] observed remarkable similarities in the protein patterns of L. obliqua cryosecretion, hemolymph, bristle extract, and tegument extract. The presence of Losac in other secretions of L. obliqua still remains to be identified. We have demonstrated here that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. The mechanism is not clear, but it is possible that t-PA and NO play an active role in Losac-induced cell survival, since the release of t-PA and NO may inhibit apoptosis and induce endothelial cell proliferation [17–19,27,28]. Losac has potential biotechnological applications and can be use to minimize cell death and consequently increase the productivity in animal cell culture, an application already explored using hemolymph of L. obliqua [10–12]. Knowledge of the structure of Losac will allow to understand its function and its mechanism of action on endothelial cells. Acknowledgments This work was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil. We thank Dr. Roberto H.P. Moraes from the Parasitology Laboratory of Butantan Institute, Sa˜o Paulo, Brazil, for the preparation of bristle extract, and Dr. Jorge Mario C.F. Junior from the Immunochemistry and Flow Cytometry Laboratory of Butantan Institute for help with some experiments with cells. We also thank the Obstetric Center of Hospital de Sa˜o Paulo (UNIFESP), Brazil, for human umbilical veins. References [1] E.M.A. Kelen, Z.P. Picarelli, A.C. Duarte, Hemorrhagic syndrome induced by contact with caterpillars of the genus Lonomia (Saturniidae, Hemileucinae), J. Toxicol. Toxin. Ver. 14 (1995) 283–308. [2] M. Zannin, D.M. Lourenc¸o, G. Motta, L.R. Dalla Costa, M. Grando, G.P. Gamborgi, M.A. Noguti, A.M. Chudzinski-Tavassi, 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 (2003) 355–364. [3] J.L. Donato, R.A. Moreno, S. Hyslop, A. Duarte, E. Antunes, B.F. Le Bonniec, F. Rendu, G. de Nucci, Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin, Thromb. Haemost. 79 (1998) 539–542. [4] C.V. Reis, E.M. Kelen, S.H.P. Farsky, F.C.V. Portaro, C.A.M. Sampaio, B.L. Fernandes, A.C.M. Camargo, A.M. ChudzinskiTavassi, A Ca2+ activated serine protease (LOPAP) could be responsible for the haemorrhagic syndrome caused by the caterpillar Lonomia obliqua, Lancet 353 (1999) 1942.

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