ARTICLE

Anticoagulant and factor Xa-like activities of Tityus discrepans scorpion venom

 

Brazón, Josmary¹*; Guerrero, Belsy²; D´Suze¹, Gina; Sevcik, Carlos¹; Arocha-Piñango, Carmen L²

¹Laboratorio de Neurofarmacología Celular, Centro de Biofísica y Bioquímica (CBB).
²Laboratorio de Fisiopatología Sección Coagulación, Centro de Medicina Experimental. Instituto Venezolano de Investigaciones Científcas (IVIC). Apartado 20632 Caracas, 1020-A. Venezuela. *jbrazon@ivic.gob.ve, jbrazon@gmail.com

Recibido: 28 de noviembre de 2012
Aceptado: 23 de abril de 2013

 

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Abstract. Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).

Keywords: Scorpion; Coagulation; Fibrinogen; Factor Xa.

Resumen. El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).

Palabras claves: Escorpión; Coagulación; Fibrinógeno; Factor Xa.

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INTRODUCTION

Many animal venoms act on the human hemostatic system as procoagulants or anticoagu-lants. The procoagulant components cause activation of the coagulation system and can induce a massive consumption of coagulant factors (Markland 1998a; 1998b; Arocha-Piñango et al. 1999a; 1999b; Reis et al. 2001; Flores et al. 2006); also some components promote pro-coagulant response of platelets by GPVI receptor activation such as Tityus discrepans venom (TdV) (Brazón et al. 2011). However, anticoagulant components may in-hibit coagulant enzymes such as factor Xa or thrombin (Markward 1994; Baskova and Za-valova 2001; Basanova et al. 2002; Motoya-shiki et al. 2003). Venoms may also cause an anticoagulant effect due to fbrinogen or fbrin degradation related with serine- or metallo-proteases (Markland 1998a; 1998b; Pinto et al. 2004; Serrano and Maroun 2005; Swenson and Markland 2005).

Tityus discrepans venom contains anticoagu-lant and procoagulant components which produce alterations of the partial thromboplastin time (PTT) and prothrombin time (PT) (Brazón et al. 2008). Furthermore, discreplasminin an antifbrinolytic compound has been isolated from TdV (Brazón et al. 2009). The purpose of this study is to describe the initial charac-terization of procoagulant and anticoagulant components from TdV. In this paper, we evidenced the presence of procoagulant com-ponents showing factor Xa-like activity, while the anticoagulant components inhibited the factor Xa activity and degraded the fbrinogen molecule.

MATERIALS AND METHODS

Animals, venom and purifcation Tityus discrepans scorpions were collected in the areas surrounding Caracas, Venezuela. Adult scorpions (~100) were kept alive in a laboratory with food and water ad libitum. Venom was extracted from them once a month. The animals were immobilized with CO₂ and the venom extraction was by electrical stimulation with pulse 50 V at 60 Hz for 100 milliseconds. The venom was dissolved in double distilled water and centrifuged at 15,000 g for 15 min at 4 °C to remove the insoluble materials. The supernatant was lyophilized and stored at - 80 °C until used. The protein concentration was estimated by spectrophotometry assuming that 1 unit of absorbance/cm of path length at 280 nm corresponds to 1 mg protein/ml (D´Suze et al. 1996). This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column (1x200 cm) equilibrated and eluted with 20 mM ammonium ac-etate, pH 4.7, with a fow rate of 0.25 ml/min. Proteins were detected at 280 nm.

Residual coagulant activity of fbrinogen pre-treated with TdV or fraction S1, was determi-nate by the thrombin time method (TT) (Austen and Rhymes 1975). Briefy, a mixture of 0.1 ml of 0.05 M Tris-HCl buffer, pH 7.4 (coagulation buffer) plus 0.1 ml of 0.3 % human fbrinogen (in coagulation buffer) treated or not with ven-om or fraction S1 for 24 h at 37°C and at a 1/30 ratio, was incubated in a borosilicate tube (10x75 mm). Then, 0.1 ml of thrombin solution (in coagulation buffer-2.5 IU/ml) was added, thoroughly mixed at 37°C and clotting time re-corded in seconds. Tests were performed three times and the median clotting time calculated. The results were expressed in seconds. Anticoagulant effect of Tityus discrepans also was evaluated using factor Xa or thrombin as substrates. The enzymes, factor Xa (0.3 nKcat/ µl) or thrombin (0.01 IU/ml) were incubated at 37 oC for 30 min with different concentrations of TdV, S1 to S5 fractions. Then, the amido-lytic activity at 405 nm was evaluated follow-ing the method of Guerrero et al. (1992), using S-2222 or S-2238 chromogenic substrate (0.8 mM and 0.6 mM, fnal concentration), respec-tively. The activity was expressed as UA/min/ µg. The enzymes incubated with buffer were used as positive controls.

Procoagulant activity

Tityus discrepans venom coagulant activity was evaluated using S-2222 and S-2238 chro-mogenic substrates, which determined the factor Xa or thrombin-like activities. A mixture of 80 µl of 50 mM Tris-HCl buffer, pH 8.4, 10 µl of sample venom (1 - 10 µg/µl) and 10 µl of substrate (0.8 mM S-2222 or 0.6 mM S-2238, fnal concentration) were placed in each well of 96 well polystyrene plates. After incubating at 37 ^oC for 30 min, the absorbance at 405 nm was measured. One unit of amidolytic activ-ity was expressed as UA/min/µg. Furthermore, factor Xa-like amidolytic activity was assayed in the presence of protease inhibitors. Prior to the assay, TdV or its fractions were incubated for 30 min at 37 °C, with each protease inhibi-tors using 10 mM 1,10 phenanthroline, 10 mM ethylenediaminetetrAαcetic acid (EDTA-Na), or 10 mM bis(2-aminoethyl) tetrAαcetic acid (EG-TA-Na) as metalloprotease inhibitors; 10 mM benzamidine, 1 µg/µl soybean trypsin inhibitor (SBTI) or 100 UI/ml aprotinin as serine prote-ase inhibitors; and 0.1 mM iodoacetic acid as cysteine protease inhibitors.

Anticoagulant activity The fbrinogenolytic activity described by Sala-zar et al. (2007) was used to determine the an-ticoagulant activity of crude venom and frac-tions. The human fbrinogen solution (in 0.05 M imidazole-0.15 M NaCl, pH 7.4 buffer) was incubated with TdV for 24 h at 37 °C, at 0.1/30; 0.25/30; 0.5/30; 1/30 and 5/30 µg venom/µg fbrinogen ratios. Fibrinogen degradation (mol-ecule or chains) was visualized by SDS-PAGE under non-reducing or reducing conditions, respectively, using Tris-Tricine-system on 5 and 7.5% gel (with 49.5% acrylamide/3% bis-acrylamide) (Schagger and Jagow 1987).

Statistical analysis

Data were processed using nonparametric statistical procedures. Data were expressed as medians and their 90% confdence intervals (Cl) with n=3, calculated by Hollander and Wolfe 1973.

RESULTS

Tityus discrepans venom was fractionated on Sephadex G-50 column and the eluted material was collected as fve fractions called S1 to S5 (Figure 1A).

The proteases present in TdV induced a slight Increase of fbrinogen electrophoretic mobilIty when a Tc/V/fbrinogen ratio ≥ 0.5/30 was used; this effect was greater at a ratios ≥1/30. At the same conditions was also evidenced a fibrinogen Aα-chains degradation; whereas Bβ and γ chains were unaffected (not shown). The ratio 1/30 was used to evalúate the effect of TdV on fbrinogen molecule/chains at different Incubation times. An increase in electrophoretic mobility of fbrinogen at times ≥16 h was observed. Fibrinogen Aα chains degradation started after 2 h of Incubation with TdV ano the effect was completed 24 h later. The Bβ and γ chains degradation did not was observed in these conditions (not shown). Fibrinogen ¡ncubation for 24 h at 37 °C with TdV or with Sephadex G-50 fraction S1 (ratio 1/30), under non reducing conditions produced an increase in fibrinogen electrophoretic mobility (Figure 1B, lañes 2 and 3). Under reducing conditions, TdV only degraded fbrinogen Aα-chains, however fraction S1 degraded both Aα and Bβ chains, generating degradation fragments with molecular weights < 31 kDa (Figure 1C, lañes 2 and 3). S2 to S5 fractions had no effect on fbrinogen molecule or chains (Figure 1B and 1C). The fbrinogen coagulant activity residual after treating with either TdV or fraction S1 (at a 1/30 ratio) was evaluated using the TT. The results showed that TT is prolonged as the fbrinogen incubation time with TdV or fraction S1 increases (not shown). In relation to factor Xa or thrombin amidolytic activity, the results showed that hundred micrograms TdV increased the factor Xa activity (0.3 nKcat/ul) from 758.70 to 864.93 (864.92-864.96) UA/min/µg and this effect was more pronounced with fraction S1 (100 µg), observIng an amidolytic activity increase from 758.70 to 998.73 (998.71- 998.77). In contrast, fractions S4 and S5 inhibited the factor Xa activity in 56.11 and 16.17%, respectively (Table 1). TdV did not modify the thrombin amidolytic activity (data not shown). These results suggested the existence in TdV of a factor Xa-like activity and a factor Xa inhibitory activity which are present in the fraction S1 (procoagulant activity) and S4 and S5 (anticoagulant activity). The results also evidenced a direct amidolytic activity with TdV due to 10 µg crude venom hydrolyzed S-2222 substrate with an amidolytic activity of 19.80 (19.79-19.81) UA/min/ µg (Table 1). This effect was more pronounced with 100 µg crude venom and the activity was of 106.23 (106.23 - 106.24) UA/min/µg. Fur-thermore, the amidolytic activity was enriched four times in fraction S1 (Table 1). Fractions S2 - S5 had no hydrolyzed the S-2222 substrate (Table 1). This amidolytic activity was reduced by > 58% in presence of serine protease inhibitors such as benzamidine or SBTI (factor Xa specifc inhibitor) (Table 1). In contrast, EDTA, EGTA, phenanthroline, iodoacetic acid or aprotinin did not modify this activity (data not shown).

Figure 1. Tityus discrepans venom (TdV) chromatography and fbrinogenolytic activity of TdV and its Sephadex G-50 fractions. (A) 7W(100 mg/ mi) was passed through a Ssphadsx G-50 column (1 x 200 cm), with 20 mM of ammonium acétate, pH 4.7, detecting at OD 280 nm with a 0.25 ml/min fow rate. Five fractions were obtained (S1-S5). (B) Fibrinogen molecule degradation (30 µg) by TdV (1 ug) or by its fractions (1 µg) after 24 h incubation at 37 °C was visualized by SDS-PAGE using Tris-Tricine-system on 5% gel under non reducing conditions. (C) Fibrinogen chains degradation (30 µg) by TdV (1 µg) or by its fractions (1 µg) after 24 h incubation at 37 °C was visualized by SDS-PAGE using Tris-Tricine-system on 7.5% gel under reducing conditions. 1.- Fibrinogen (Fg) control. 2.- Fg + TdV. 3.- Fg + S1. 4.- Fg + S2. Fg + S3. Fg + S4. Fg + S5. Fibrinogen consists of three polypeptide chains Aα, BB and y. Standard proteins were used as a reference to determine the molecular weight. The gels were stained with R-250 Coomassie Blue.

Table 1. Factor Xa-like and Factor Xa inhibitory activities of TdV and its Sephadex G-50 fractions.
Factor Xa-like activity was measured by an amidolytic micromethod (S-2222 substrate) in presence or absence of protease inhibitors. To evalúate the effect of TdV or its fraction on factor Xa activity was incubated for 30 min at 37°C factor Xa (0.3 nkat/ul) with TdV or Sephadex-G50 fractions. Data were expressed as median and its IC at 90% confidence, n = 3, ND = No determined. Benz: Benzamidine.

 

In addition, nor TdV neither the chromatographic fractions showed thrombin-like amidolytic activity.

DISCUSSION

Scorpions using their venom to defend themselves from predators or to capture their prey; their venoms are complex mixtures of biologi-cally active components over different types of organisms, such as bacteria, insects, fungi and mammals (D'Suze et al. 2004a; 2004b; Díaz et al. 2009; Joya et al. 2011). Scorpion venoms are generally not known to have the ability to produce hemostatic alterations. However, there are exceptions as Centruroides sculpturatus (Watt et al., 1974; Longenecker and Longenecker 1981), Buthus tamulus (Devi et al. 1970; Reddy et al. 1972), Buthus martensii (Song et al. 2005) and Tityus discrepans (D´Suze et al. 2003; 2004b; Brazón et al. 2008; 2009; 2011) that their venoms, in case of se-vere envenoming perturb the mammal hemostatic system. The relevance of components with action on haemostatic system (platelet, coagulation and fbrinolysis) in scorpion venoms is not clear. However, the presence of these components has pathophysiologic im-plications severe in the scorpion envenoming. The results of fbrinogenolytic activity indicate that TdV contains fbrinogenases that induce fbrinogen degradation associated with an an-ticoagulant effect. Similar components were isolated from snake venoms, such as ancrod, this compound is medically important as ther-apeutic agents (Swenson and Markland 2005). On the other hand, the results of this study indicated the presence of anticoagulant components with factor Xa inhibitory activity in TdV, which are responsible of the previously described PTT and PT prolongation (Brazón et al. 2008). Furthermore, this venom produces a factor Xa-like activity (procoagulant activity) which was found in fraction S1; this fnding is con-sistent with the synergism in amidolytic activ-ity observed when commercial factor Xa was incubated with either TdV or fraction S1. Prob-ably, the components with the factor Xa-like activity present in the venom and S1 induced the previously described PTT shortening (Brazón et al. 2008). The benzamidine or SBTI effects on the venom amidolytic activity, supports the notion that a serineproteases with factor Xa-like activity exists in T. discrepans venom. Additionally, the TdV did not hydrolyze the S-2238 substrate. These results indicate the absence of thrombin-like activity in venom (data not shown).

We have shown that TdV, in addition to t-PA-like compounds and discreplasminin, a plas-min inhibitor (Brazón et al. 2009), also contains anticoagulant compounds able to inhibit factor Xa amidolytic activity and to induce fbrinogen degradation. Our results also demonstrated the existence of a procoagulant activity which was similar to factor Xa. All these compounds, per se or in combination, could explain the al-terations of PT and PTT (Brazón et al. 2008) as well as the fbrin deposits observed in lung alveoli from experimentally envenomed mam-mals (D'Suze et al. 2004b). The most dangerous complication induced by TdV is the "scorpion venom respiratory distress syndrome (SVRDS)". Our group has demonstrated that this complication started by neutrophil lung infltration and sequestra-tion, with important fbrin deposition as the last step of a cascade reaction where leuko-cytes activation products are involved (D'Suze et al. 2003). However the pathophysiology of SVRDS is complex and cannot be entirely at-tributed to the unregulated leukocytes activa-tion products, SVRDS may in part be associ-ated with the procoagulant components found in TdV. These are not abundant, < 1% of the venom, but are able to produce coagulation disorders both in scorpionism patients as well as in vitro studies (Brazón et al. 2008; D'Suze et al. 2003, 2004). Perhaps venom procoagu-lant components act in a synergistic form with neutrophil procoagulant products enhancing fbrin clots formation. These critical questions wait for answers from ongoing studies. This investigation has motivated the further purifcation and physiological characterization of the compounds responsible of the haemo-static alterations reported here. These com-pounds are very interesting due its biotechno-logical applications on clinical disorders asso-ciated with the fbrinolytic system and coagu-lation, it is mandatory to determine their action mechanisms, physiological effects and phar-macokinetic parameters.

Acknowledgements

The authors are indebted to the people of San Antonio de Los Altos, Miranda, Venezuela and their Fire Department for the supply of scorpions. We thank to Moisés Sandoval for the technical assistance. We are grateful with Zoila Carvajal and Ana Maria Salazar for the tech-nical assistance as for their advice and com-ments. This research was supported by grants from FONACIT and IVIC (Caracas, Venezuela).

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