316 ISSN 1607 6729, Doklady Biochemistry and Biophysics, 2017, Vol 476, pp 316–319 © Pleiades Publishing, Ltd , 2017 Original Russian Text © Tran Vu Thien, Hoang Ngoc Anh, Nguyen Thi Thuy Trang, Phung[.]
ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2017, Vol 476, pp 316–319 © Pleiades Publishing, Ltd., 2017 Original Russian Text © Tran Vu Thien, Hoang Ngoc Anh, Nguyen Thi Thuy Trang, Phung Van Trung, Nguyen Cuu Khoa, A.V Osipov, P.V Dubovskii, I.A Ivanov, A.S Arseniev, V.I Tsetlin, Yu.N Utkin, 2017, published in Doklady Akademii Nauk, 2017, Vol 476, No 4, pp 476–479 BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY Low-Molecular-Weight Compounds with Anticoagulant Activity from the Scorpion Heterometrus laoticus Venom Tran Vu Thiena, b, Hoang Ngoc Anha, Nguyen Thi Thuy Trang c, Phung Van Trungd, Nguyen Cuu Khoaa, A V Osipove, P V Dubovskiie, I A Ivanovd, A S Arsenieve, Corresponding Member of the RAS V I Tsetline, and Yu N Utkine * Received April 26, 2017 Abstract—Low-molecular-weight compounds with anticoagulant activity were isolated from the scorpion Heterometrus laoticus venom The determination of the structure of the isolated compounds by nuclear magnetic resonance and mass spectrometry showed that one of the isolated compounds is adenosine, and the other two are dipeptides leucyl-tryptophan and isoleucyl-tryptophan The anticoagulant properties of adenosine, which is an inhibitor of platelet aggregation, is well known, but its presence in scorpion venom is shown for the first time The ability of leucyl-tryptophan and isoleucyl-tryptophan to slow down blood clotting and their presence in scorpion venom are also established for the first time DOI: 10.1134/S1607672917050052 Scorpion venom is a complex mixture of compounds of polypeptide nature It exhibits primarily neurotoxic effects and instantly paralyzes small prey Bites of large tropical scorpions may be fatal to humans; in this case, the symptoms of the nervous system damage are mostly observed However, some scorpion venoms cause blood clotting disorders In particular, Pandinus imperator and Parabuthus transvaalicus venoms increase the blood clotting time 2.5 and 2.3 times, respectively, whereas other venoms increase this value 0.8–2 times [1] Recently it was shown that the Tityus discrepans venom and its fractions obtained by gel filtration affect the blood clotting parameters [2] The group of peptides that affect blood clotting include the toxin isolated from the Chinese scorpion Buthus martensii Karsch venom It belongs to the group of scorpion venom active polypeptides (SVAP) and induces platelet aggregation and thrombus formation in rabbits in vivo and in vitro as well as changes in the blood plasma levels of thromboxane B2 a Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam b Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Vietnam c Faculty of Pharmacy, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam d Istitute of Chemical Technology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam e Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia *e-mail: utkin@mx.ibch.ru, yutkin@yandex.ru and 6-keto prostaglandin F1-alpha This polypeptide is the main component of the scorpion venom [3] Recently, the peptide discreplasminin, which was isolated from the scorpion Tityus discrepans and is a plasmin inhibitor, has been described It has a molecular mass of approximately kDa and, similarly to aprotinin, exhibits antifibrinolytic activity It is also assumed that it interacts with the plasmin active site and tissue plasminogen activator [4] Earlier, we found that fractions of the scorpion Heterometrus laoticus venom, obtained by gel filtration, affect blood clotting processes [5] In the present study, we have identified in this venom low-molecular-weight compounds increasing the blood clotting time and have established their structure The anticoagulant activity in vitro was determined by the standard coagulometric tests: activated partial thromboplastin time (APTT), prothrombin (thromboplastin) time (PTT), and thrombin time (TT) The anticoagulant activity in vivo was determined by the change in the bleeding time of mouse tail wounds [6] For this purpose, the mouse tail tip approximately mm long was cut off with scissors (tail diameter, approximately 1.5 mm), the tail was immersed into a vessel with 0.9% NaCl at 37°C, and the bleeding time (from the beginning until complete end) was determined As was shown previously [5], the anticoagulant activity was exhibited by the low-molecular-weight fraction 5, obtained by gel filtration of the H laoticus crude venom on a Sephadex G-50 column [5, 7] To isolate the compounds with the anticoagulant activity, fraction was subjected to further separation by reversed-phase chromatography, and the effect of the 316 LOW-MOLECULAR-WEIGHT COMPOUNDS Aromatics-Trp αH-Trp 317 δ1, 2CH3-Leu β1, 2H-Trp β1H-Leu β2H-Leu αH-Leu γH-Leu ppm 0.72 2.81 3.00 0.81 0.75 0.88 0.96 0.88 0.87 0.78 0.67 1.54 0.83 Fig Proton nuclear magnetic resonance spectrum for the compound from fraction 5-21 obtained fractions on the bleeding time in mice was analyzed It was found that, out of the 24 fractions obtained, six fractions to some extent increased the bleeding time Three of these six fractions (5-5, 5-21, and 5-2) were further purified by reversed-phase chromatography, and their structure was analyzed by nuclear magnetic resonance and mass spectrometry Mass spectra were recorded using a LCQ DECA XP+ mass spectrometer (Thermo Finnigan, United States) According to mass spectrometry, the mass of the compound from fraction 5-5 was 267.8 Da, and the compounds from fractions 5-21 and 5-22 had an identical mass of 317.1 Da Since the mass of the product from fraction 5-5 was close to the mass of adenosine (267.2 Da), we performed a chromatographic experiment with a simultaneous loading of these two compounds on a reversed-phase chromatographic column As a result, both compounds were eluted from the column as a single peak, which was analyzed by mass spectrometry The resulting mass spectrum was identical to that for the compound from fraction 5-5 These data suggest that fraction 5-5 contained adenosine The compound from fraction 5-21 was analyzed by proton nuclear magnetic resonance in D2O using a DRX-500 spectrometer (Bruker Corp., United States) with an operating frequency for hydrogen of 500.13 MHz The spectrum was recorded in D2O in a single-pulse mode with a spectral capture width of DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol 476 10 ppm The spectral capture width in H2O was 16 ppm The spectrum of the obtained compound is shown in Fig In the spectrum, spin systems of two amino acid residues corresponding to leucine and tryptophan residues were identified However, the spectrum in D2O did not allow us to determine the sequence of amino acids: Leu–Trp or Trp–Leu To resolve this ambiguity, the spectrum in H2O was recorded under conditions described in [8] The sequence of amino acid residues was determined by splitting the alpha-proton signals A multiplet in the tryptophan residue was found A narrow multiplet was also detected in leucine When this peptide was transferred to H2O, this signal did not experience additional splitting from the amide proton, whereas the alphaproton of tryptophan experienced it This fact suggests that we deal with the Leu–Trp sequence, because the proton of the alpha-amino group of leucine was in the NH3+ form and did not have the constant of the spin– spin interaction with its alpha-proton Thus, the structure of the analyzed peptide was Leu–Trp The analysis of the compound from fraction 5-22 by tandem mass spectrometry showed that this is a dipeptide consisting of leucine/isoleucine and tryptophan residues, with tryptophan being the C-terminal residue Since the Leu–Trp dipeptide was present in fraction 5-21, it was assumed that fraction 5-22 contained the Ile–Trp dipeptide To test this hypothesis, 2017 318 THIEN et al Table Duration of bleeding from the tail wound of mice after the injection of test compounds Time elapsed after sample injection Sample 20 30 60 90 120 Bleeding time, s Control Adenosine Leu–Trp Ile–Trp 79.5 ± 13.7 248.2 ± 66.7* 314.5 ± 85.2* 233.0 ± 30.6** 43.33 ± 1.94 314 ± 58.6* 84.8 ± 16.7 179.0 ± 41.4* 45.83 ± 3.95 146.7 ± 46.0* 81.2 ± 15.4 218.7 ± 78.5** 40.67 ± 5.02 65 ± 14.5 61.8 ± 14.8 151.5 ± 57.4 49.67 ± 7.85 40.2 ± 10.3 68.8 ± 16.4 83.8 ± 13.7 Data are represented as M ± m, n = for each group * p < 0.05 and ** p < 0.01 compared to the control we synthesized both dipeptides When comparing the structures of the synthetic and natural peptides by reversed-phase chromatography and mass spectroscopy, we found that fraction 5-22 contained the Ile– Trp dipeptide It should be noted that data on the detection of adenosine or Leu–Trp and Ile–Trp dipeptides in scorpion venoms are missing in available literature Thus, we were the first to detect these compounds in scorpion venom To study the anticoagulant activity, solutions of synthetic dipeptides and adenosine in 0.9% NaCl were injected into the lateral tail vein of mice at a dose of 2.48 mg/kg (injection volume, 0.1 mL per 10 gram of mouse body weight) Mice of the control group was injected with 0.9% NaCl The significance of differences between the groups was estimated by the Kruskal–Wallis test using the Minitab 15.0 software (Minitab Inc., United States) The results of these experiments are shown in Table Adenosine and dipeptides in varying degrees prolonged the bleeding time, i.e., exhibited anticoagulant activity Adenosine increased the bleeding time relative to the control group throughout the observation time (120 min) These differences were significant in the first hour of observations and nonsignificant in the second hour Similarly, the Ile–Trp dipeptide reliably and significantly prolonged the bleeding time in the first hour after injection The least activity was detected for the Leu–Trp dipeptide, which significantly increased the bleeding time only during the first 20 after injection It should be noted that we detected no anticoagulant activity of these dipeptide at concentrations up to 100 µM in the standard coagulometric tests APTT, PTT, and TT Endogenous adenosine modulates many physiological processes and functions, in particular, as a strong antiinflammatory agent and an inhibitory neurotransmitter Published data indicate that adenosine also exhibits antiplatelet activity For example, adenosine at submicromolar concentrations exerts an anticoagulant effect on human whole blood [9] Since adenosine has an extremely short lifetime in blood plasma [10], to detect its effect we used it at a sufficiently high dose (2.48 mg/kg) At this dose, the observed effect was sufficiently long, and an increased bleeding time (compared to the control) was observed for h after the adenosine injection (Table 1) Interestingly, at the same dose, a stronger effect was exerted by the Ile–Trp dipeptide: an increased bleeding time was observed for h after its injection, although the differences were not statistically significant during the second hour (Table 1) The observed effect is the first indication that the dipeptides can increase the blood clotting time in vivo Given the lack of effects in APTT, PTT, and TT tests in vitro, it can be expected that dipeptides inhibit platelet aggregation It should be noted that the tryptophan-containing dipeptides, including Ile–Trp and Leu–Trp, which were found in food protein hydrolysates, are inhibitors of the angiotensin-converting enzyme, which is involved in the blood pressure regulation [11] The dipeptide Ile–Trp is a selective and competitive inhibitor of the C-terminal domain of the enzyme with a selectivity coefficient of 40 as compared to the N-terminal domain [11] There are also evidences that the Ile–Trp under the BNC210 trademark (Bionomics Limited, Australia) passes the second stage of clinical trials for the treatment of post-traumatic stress disorder [12] According to the mechanism of action, BNC210 is a negative allosteric modulator of nicotinic acetylcholine receptors of α7 type [13] Given the fact that platelet aggregation is substantially inhibited [14] by α-bungarotoxin and methyllycaconitine, which are selective antagonists of nicotinic acetylcholine receptors of α7 type, it can be suggested that the observed anticoagulant effect of the Ile–Trp dipeptide is mediated by its interaction with this receptor To conclude, this is the first study to show the presence of adenosine and Leu–Trp and Ile–Trp dipeptides in a scorpion venom We are also the first to show that Leu–Trp and Ile–Trp dipeptides exhibit anticoagulant activity in vivo, increasing the bleeding time in mice ACKNOWLEDGMENTS The study was supported by the Russian Science Foundation (project no 16-14-00215) DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol 476 2017 LOW-MOLECULAR-WEIGHT COMPOUNDS REFERENCES Tan, N.H and Ponnudurai, G., Comparative study of the enzymatic, hemorrhagic, procoagulant and anticoagulant activities of some animal venoms, Comp Biochem Physiol C: Comp Pharmacol., 1992, vol 103, no 2, pp 299–302 Brazon, J., Guerrero, B., D’Suze, G., Sevcik, C., and Arocha-Pinango, C.L., Anticoagulant and factor Xalike activities of Tityus discrepans scorpion venom, Acta Toxicol Argent, 2013, vol 21, pp 26–32 Song, Y.M., Tang, X.X., Chen, X.G., Gao, B.B., Gao, E., Bai, L., and Lv, X.R., Effects of scorpion venom bioactive polypolypeptides on platelet aggregation and thrombosis and plasma 6-keto-PG F1α and TXB2 in rabbits and rats, Toxicon, 2005, vol 46, no 2, pp 230– 235 Brazón, J., D’Suze, G., D’Errico, M.L., ArochaPiñango, C.L., and Guerrero, B., Discreplasminin, a plasmin inhibitor isolated from Tityus discrepans scorpion venom, Arch Toxicol., 2009, vol 83, no 7, pp 669–678 Hoang, N.A., Vo, D.M.H., Nikitin, I., and Utkin, Y., Isolation and preliminary study of short toxins from scorpion Heterometrus laoticus, Tap Chi Hoa Hoc (J Chem.), 2011, vol 49, pp 118–122 Liu, Y., 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by M Batrukova 2017 ... AND BIOPHYSICS Vol 476 2017 LOW- MOLECULAR- WEIGHT COMPOUNDS REFERENCES Tan, N.H and Ponnudurai, G., Comparative study of the enzymatic, hemorrhagic, procoagulant and anticoagulant activities of.. .LOW- MOLECULAR- WEIGHT COMPOUNDS Aromatics-Trp αH-Trp 317 δ1, 2CH3-Leu β1, 2H-Trp β1H-Leu β2H-Leu αH-Leu γH-Leu... performed a chromatographic experiment with a simultaneous loading of these two compounds on a reversed-phase chromatographic column As a result, both compounds were eluted from the column as