Back
 AE  Vol.9 No.1 , January 2021
Partial Characterization of Thrombin Inhibitor(s) Derived from Salivary Glands of the Tick, Hyalomma dromedarii, and Related Anti-Cancer Potential
Abstract: A long-term blood feeder, like the Hyalomma dromedarii tick, requires extended control over all hemostatic defense mechanisms generated by the host during feeding, including blood coagulation. To overcome this, ticks have evolved numerous molecules that target proteases in the blood coagulation cascade. New insights into the role of clotting factors in the development and progression of cancer have identified anticoagulant treatment as a potential therapeutic approach. In this context, the present work assessed the anticoagulation activities of crude and fractionated salivary gland extract (SGE) prepared from semi-fed H. dromedarii females. Additionally, the antitumor effects of the potent anti-thrombin fractions were determined against colon cancer (Caco-2) and normal skin (HFB4) cells. Crude SGE significantly prolonged clotting time in prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) assays and inhibited thrombin in FII-activity assay. Using anion-exchange chromatography, the fractions that strongly inhibited thrombin (3.A4 and 3.A5) were eluted. Both fractions prolonged the aPTT and TT clotting times and reduced the activity of FII significantly. The protein profiles of both fractions indicated the presence of a single polypeptide band of about 99 kDa. Regarding anti-cancer potential of the tested fractions, Caco-2 cells showed reduced viability with obvious morphological changes, induced apoptosis and a reduced level of vascular endothelial growth factor (VEGF). G2/M cell cycle arrest was observed only in 3.A5-treated cells. No cytotoxic effects were observed in HFB4 cells. These results demonstrated the potential of tick-derived anticoagulants, specifically thrombin inhibitors, as effective tools in colorectal cancer treatment. Further purification of the effector molecule(s) is required to fully characterize their structures and mechanisms of action.
Cite this paper: Ibrahim, W. , Mohamed, F. , Moselhy, W. , Samie, E. , Mohamed, A. (2021) Partial Characterization of Thrombin Inhibitor(s) Derived from Salivary Glands of the Tick, Hyalomma dromedarii, and Related Anti-Cancer Potential. Advances in Entomology, 9, 1-19. doi: 10.4236/ae.2021.91001.
References

[1]   Abdullah, H.H.A.M., El-Shanawany, E.E., Abdel-Shafy, S., Abou-Zeina, H.A.A. and Abdel-Rahman, E.H. (2018) Molecular and Immunological Characterization of Hyalomma dromedarii and Hyalomma excavatum (Acari: Ixodidae) Vectors of Q Fever in Camels. Veterinary World, 11, 1109-1119.
https://doi.org/10.14202/vetworld.2018.1109-1119

[2]   Hoffman, R., Benz, E.J., Furie, B. and Shattil, S.J. (2009) Hematology: Basic Principles and Practice. Churchill Livingstone/Elsevier, Edinburgh, New York.

[3]   Chudzinski-Tavassi, A.M., Faria, F. and Flores, M.P.A. (2018) Anticoagulants from Hematophagous. In: Božič-Mijovski, M., Ed., Anticoagulant Drugs, IntechOpen, London, 39-68.
https://doi.org/10.5772/intechopen.78025

[4]   Stibrániová, I., Bartíková, P., Holíková, V. and Kazimírová, M. (2019) Deciphering Biological Processes at the Tick-Host Interface Opens New Strategies for Treatment of Human Diseases. Frontiers in Physiology, 10, 830.
https://doi.org/10.3389/fphys.2019.00830

[5]   Blisnick, A.A., Foulon, T. and Bonnet, S.I. (2017) Serine Protease Inhibitors in Ticks: An Overview of Their Role in Tick Biology and Tick-Borne Pathogen Transmission. Frontiers in Cellular and Infection Microbiology, 7, 199.
https://doi.org/10.3389/fcimb.2017.00199

[6]   Crawley, J.T.B., Zanardelli, S., Chion, C.K.N.K. and Lane, D.A. (2007) The Central Role of Thrombin in Hemostasis. Journal of Thrombosis and Haemostasis, 5, 95-101.
https://doi.org/10.1111/j.1538-7836.2007.02500.x

[7]   Keragala, C.B., Draxler, D.F., mcQuilten, Z.K. and Medcalf, R.L. (2018) Haemostasis and Innate Immunity—A Complementary Relationship: A Review of the Intricate Relationship between Coagulation and Complement Pathways. British Journal of Haematology, 180, 782-798.
https://doi.org/10.1111/bjh.15062

[8]   Nuttall, P.A. (2019) Wonders of Tick Saliva. Ticks and Tick-Borne Diseases, 10, 470-481.
https://doi.org/10.1016/j.ttbdis.2018.11.005

[9]   Falanga, A., Russo, L., Milesi, V. and Vignoli, A. (2017) Mechanisms and Risk Factors of Thrombosis in Cancer. Critical Reviews in Oncology/Hematology, 118, 79-83.
https://doi.org/10.1016/j.critrevonc.2017.08.003

[10]   Degen, J.L. and Palumbo, J.S. (2012) Hemostatic Factors, Innate Immunity and Malignancy. Thrombosis Research, 129, S1-S5.
https://doi.org/10.1016/S0049-3848(12)70143-3

[11]   Bobek, V. and Kovarik, J. (2004) Antitumor and Antimetastatic Effect of Warfarin and Heparins. Biomedicine and Pharmacotherapy, 58, 213-219.
https://doi.org/10.1016/j.biopha.2003.11.007

[12]   Zalpour, A., Kroll, M.H., Afshar-Kharghan, V., Yusuf, S.W. and Escalante, C. (2011) Role of Factor Xa Inhibitors in Cancer-Associated Thrombosis: Any New Data? Advances in Hematology, 2011, Article ID: 196135.
https://doi.org/10.1155/2011/196135

[13]   Featherby, S., Xiao, Y.P., Ettelaie, C., Nikitenko, L.L., Greenman, J. and Maraveyas, A. (2019) Low Molecular Weight Heparin and Direct Oral Anticoagulants Influence Tumour Formation, Growth, Invasion and Vascularisation by Separate Mechanisms. Scientific Reports, 9, Article No. 6272.
https://doi.org/10.1038/s41598-019-42738-1

[14]   Carneiro-Lobo, T.C., Konig, S., Machado, D.E., Nasciutti, L.E., Forni, M.F., Francischetti, I.M., Sogayar, M.C. and Monteiro, R.Q. (2009) Ixolaris, a Tissue Factor Inhibitor, Blocks Primary Tumor Growth and Angiogenesis in a Glioblastoma Model. Journal of Thrombosis and Haemostasis, 7, 1855-1864.
https://doi.org/10.1111/j.1538-7836.2009.03553.x

[15]   Chudzinski-Tavassi, A.M., De-Sá-Júnior, P.L., Simons, S.M., Maria, D.A., Ventura, J.S., Batista, I.F.C., Faria, F., Durães, E., Reis, E.M. and Demasi, M. (2010) A New Tick Kunitz Type Inhibitor, Amblyomin-X, Induces Tumor Cell Death by Modulating Genes Related to the Cell Cycle and Targeting the Ubiquitin-Proteasome System. Toxicon, 56, 1145-1154.
https://doi.org/10.1016/j.toxicon.2010.04.019

[16]   Estrada-Peña, A. (2004) Ticks of Domestic Animals in the Mediterranean Region: A Guide to Identification of Species. University of Zaragoza, Zaragoza.

[17]   Heller-Haupt, A., Kagaruki, L.K. and Varma, M.G.R. (1996) Resistance and Cross-Resistance in Rabbits to Adults of Three Species of African Ticks (Acari: Ixodidae). Experimental and Applied Acarology, 20, 155-165.
https://doi.org/10.1007/BF00051481

[18]   Becker, U., Jering, H. and Roschlau, P. (1984) Coagulation Methods. In: Bergmeyer, H.U., Ed., Methods of Enzymatic Analysis, Vol. 5. Academic Press, New York, 486-499.

[19]   Fickenscher, K. (1998) Thrombin Time (TT) Test. In: Lothar, T., Ed., Clinical Laboratory Diagnostics: Use and Assessment of Clinical Laboratory Results, TH-Books Verlagsgesellschaft mbH, Frankfurt/Main, 604-605.

[20]   van den Besselaar, A.M.H.P. (1991) Principles of Coagulation Factor Assays. In: Sibinga, C.T.S., Das, P.C. and Mannucci, P.M., Eds., Coagulation and Blood Transfusion, Developments in Hematology and Immunology, Vol. 26, Springer, Boston, 165-174.
https://doi.org/10.1007/978-1-4615-3900-1_16

[21]   Simons, S.M., Junior, P.L., Faria, F., Batista, I.F., Barros-Battesti, D.M., Labruna, M.B. and Chudzinski-Tavassi, A.M. (2011) The Action of Amblyomma cajennense Tick Saliva in Compounds of the Hemostatic System and Cytotoxicity in Tumor Cell Lines. Biomedicine and Pharmacotherapy, 65, 443-450.
https://doi.org/10.1016/j.biopha.2011.04.030

[22]   Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head Bacteriophage T4. Nature, 227, 680-685.
https://doi.org/10.1038/227680a0

[23]   Hansen, M.B., Nielsen, S.E. and Berg, K. (1989) Re-Examination and Further Development of a Precise and Rapid Dye Method for Measuring Cell Growth/Cell Kill. Journal of Immunological Methods, 119, 203-210.
https://doi.org/10.1016/0022-1759(89)90397-9

[24]   Šimo, L., Kazimírová, M., Richardson, J. and Bonnet, S.I. (2017) The Essential Role of Tick Salivary Glands and Saliva in Tick Feeding and Pathogen Transmission. Frontiers in Cellular and Infection Microbiology, 7, 281.
https://doi.org/10.3389/fcimb.2017.00281

[25]   Koh, C.Y., Modahl, C.M., Kulkarni, N. and Kini, R.M. (2018) Toxins Are Excellent Source of Therapeutic Agents against Cardiovascular Diseases. Seminars in Thrombosis and Hemostasis, 44, 691-706.
https://doi.org/10.1055/s-0038-1661384

[26]   Gordon, J.R. and Allen, J.R. (1991) Factors V and VII Anticoagulant Activities in the Salivary Glands of Feeding Dermacentor andersoni Ticks. Journal of Parasitology, 77, 167-170.
https://doi.org/10.2307/3282577

[27]   Tobu, M., Iqbal, O., Hoppensteadt, D.A., Shultz, C., Jeske, W. and Fareed, J. (2002) Effects of a Synthetic Factor Xa Inhibitor (JTV-803) on Various Laboratory Tests. Clinical and Applied Thrombosis/Hemostasis, 8, 325-336.
https://doi.org/10.1177/107602960200800404

[28]   Garcia, D., Barrett, Y.C., Ramacciotti, E. and Weitz, J.I. (2013) Laboratory Assessment of the Anticoagulant Effects of the Next Generation of Oral Anticoagulants. Journal of Thrombosis and Haemostasis, 11, 245-252.
https://doi.org/10.1111/jth.12096

[29]   Joubert, A.M., Crause, J.C., Gaspar, A., Clarke, F.C., Spickett, A.M. and Neitz, A.W.H. (1995) Isolation and Characterization of an Anticoagulant Present in the Salivary Glands of the Bont-Legged Tick, Hyalomma truncatum. Experimental and Applied Acarology, 19, 79-92.
https://doi.org/10.1007/BF00052548

[30]   Davie, E.W. (2003) A Brief Historical Review of the Waterfall/Cascade of Blood Coagulation. Journal of Biological Chemistry, 278, 50819-50832.
https://doi.org/10.1074/jbc.X300009200

[31]   Coughlin, S.R. (2005) Protease-Activated Receptors in Hemostasis, Thrombosis and Vascular Biology. Journal of Thrombosis and Haemostasis, 3, 1800-1814.
https://doi.org/10.1111/j.1538-7836.2005.01377.x

[32]   Bode, W. (2006) Structure and Interaction Modes of Thrombin. Blood Cells, Molecules and Diseases, 36, 122-130.
https://doi.org/10.1016/j.bcmd.2005.12.027

[33]   Weitz, J.I. (2007) Factor Xa or Thrombin: Is Thrombin a Better Target? Journal of Thrombosis and Haemostasis, 5, 65-67.
https://doi.org/10.1111/j.1538-7836.2007.02552.x

[34]   Meekins, D.A., Kanost, M.R. and Michel, K. (2016) Serpins in Arthropod Biology. Seminars in Cell and Developmental Biology, 62, 105-119.
https://doi.org/10.1016/j.semcdb.2016.09.001

[35]   Bensaoud, C., Nishiyama Jr., M.Y., Ben-Hamda, C., Lichtenstein, F., de Oliveira, U.C., Faria, F., Junqueira-de-Azevedo, I.L.M., Ghedira, K., Bouattour, A., M’Ghirbi, Y. and Chudzinski-Tavassi, A.M. (2018) De Novo Assembly and Annotation of Hyalomma dromedarii Tick (Acari: Ixodidae) Sialotranscriptome with Regard to Gender Differences in Gene Expression. Parasites & Vectors, 11, 314.
https://doi.org/10.1186/s13071-018-2874-9

[36]   Bensaoud, C., Aounallah, H., Sciani, J.M., Faria, F., Chudzinski-Tavassi, A.M., Bouattour, A. and M’Ghirbi, Y. (2019) Proteomic Informed by Transcriptomic for Salivary Glands Components of the Camel Tick Hyalomma dromedarii. BMC Genomics, 20, 675.
https://doi.org/10.1186/s12864-019-6042-1

[37]   Ibrahim, M.A. and Masoud, H.M.M. (2018) Thrombin Inhibitor from the Salivary Gland of the Camel Tick Hyalomma dromedarii. Experimental and Applied Acarology, 74, 85-97.
https://doi.org/10.1007/s10493-017-0196-9

[38]   Francischetti, I.M.B., Anderson, J.M., Manoukis, N., Pham, V.M. and Ribeiro, J.M.C. (2011) An Insight into the Sialotranscriptome and Proteome of the Coarse Bont-Legged Tick, Hyalomma marginatum rufipes. Journal of Proteomics, 74, 2892-2908.
https://doi.org/10.1016/j.jprot.2011.07.015

[39]   Jablonka, W., Kotsyfakis, M., Mizurini, D.M., Monteiro, R.Q., Lukszo, J., Drake, S.K., Ribeiro, J.M.C. and Andersen, J.F. (2015) Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin. PLoS ONE, 10, e0133991.
https://doi.org/10.1371/journal.pone.0133991

[40]   Reddel, C.J., Tan, C.W. and Chen, V.M. (2019) Thrombin Generation and Cancer: Contributors and Consequences. Cancers, 11, 100.
https://doi.org/10.3390/cancers11010100

[41]   Palumbo, J.S., Talmage, K.E., Massari, J.V., La Jeunesse, C.M., Flick, M.J., Kombrinck, K.W., Hu, Z., Barney, K.A. and Degen, J.L. (2007) Tumor Cell-Associated Tissue Factor and Circulating Hemostatic Factors Cooperate to Increase Metastatic Potential through Natural Killer Cell-Dependent and -Independent Mechanisms. Blood, 110, 133-141.
https://doi.org/10.1182/blood-2007-01-065995

[42]   Yokota, N., Zarpellon, A., Chakrabarty, S., Bogdanov, V.Y., Gruber, A., Castellino, F.J., Mackman, N., Ellies, L.G., Weiler, H., Ruggeri, Z.M. and Ruf, W. (2014) Contributions of Thrombin Targets to Tissue Factor-Dependent Metastasis in Hyperthrombotic Mice. Journal of Thrombosis and Haemostasis, 12, 71-81.
https://doi.org/10.1111/jth.12442

[43]   Adams, G.N., Rosenfeldt, L., Frederick, M., Miller, W., Waltz, D., Kombrinck, K., McElhinney, K.E., Flick, M.J., Monia, B.P., Revenko, A.S. and Palumbo, J.S. (2015) Colon Cancer Growth and Dissemination Relies upon Thrombin, Stromal PAR-1, and Fibrinogen. Cancer Research, 75, 4235-4243.
https://doi.org/10.1158/0008-5472.CAN-15-0964

[44]   Vossen, C.Y., Hoffmeister, M., Chang-Claude, J.C., Rosendaal, F.R. and Brenner, H. (2011) Clotting Factor Gene Polymorphisms and Colon Cancer Risk. Journal of Clinical Oncology, 29, 1722-1727.
https://doi.org/10.1200/JCO.2010.31.8873

[45]   Turpin, B., Miller, W., Rosenfeldt, L., Kombrinck, K., Flick, M.J., Steinbrecher, K.A., Harmel-Laws, E., Mullins, E.S., Shaw, M., Witte, D.P., Revenko, A., Monia, B. And Palumbo, J.S. (2014) Thrombin Drives Tumorigenesis in Colitis-Associated Colon Cancer. Cancer Research, 74, 3020-3030.
https://doi.org/10.1158/0008-5472.CAN-13-3276

[46]   DiPaola, R.S. (2002) To Arrest or Not to G2-M Cell-Cycle Arrest. Clinical Cancer Research, 8, 3311-3314.

[47]   Akagi, E.M., Júnior, P.L., Simons, S.M., Bellini, M.H., Barreto, S.A. and Chudzinski-Tavassi, A.M. (2012) Pro-Apoptotic Effects of Amblyomin-X in Murine Renal Cell Carcinoma “in Vitro”. Biomedicine and Pharmacotherapy, 66, 64-69.
https://doi.org/10.1016/j.biopha.2011.11.015

[48]   Morais, K.L., Pacheco, M.T., Berra, C.M., Bosch, R.V., Sciani, J.M., Chammas, R., Saito, R.F., Iqbal, A. and Chudzinski-Tavassi, A.M. (2016) Amblyomin-X Induces ER Stress, Mitochondrial Dysfunction, and Caspase Activation in Human Melanoma and Pancreatic Tumor Cell. Molecular and Cellular Biochemistry, 415, 119-131.
https://doi.org/10.1007/s11010-016-2683-4

[49]   Rmali, K.A., Puntis, M.C. and Jiang, W.G. (2007) Tumor-Associated Angiogenesis in Human Colorectal Cancer. Colorectal Disease, 9, 3-14.
https://doi.org/10.1111/j.1463-1318.2006.01089.x

[50]   Mihalache, A. and Rogoveanu, I. (2014) Angiogenesis Factors Involved in the Pathogenesis of Colorectal Cancer. Current Health Sciences Journal, 40, 5-11.

[51]   Ellis, L.M., Takahashi, Y., Liu, W. and Shaheen, R.M. (2000) Vascular Endothelial Growth Factor in Human Colon Cancer: Biology and Therapeutic Implications. Oncologist, 5, 11-15.
https://doi.org/10.1634/theoncologist.5-suppl_1-11

[52]   Abdou, A.G., Aiad, H., Asaad, N., Abd El-Wahed, M. and Serag El-Dien, M. (2006) Immunohistochemical Evaluation of Vascular Endothelial Growth Factor (VEGF) in Colorectal Carcinoma. Journal of the Egyptian National Cancer Institute, 18, 311-322.

[53]   Mohamed, S.Y., Mohammed, H.L., Ibrahim, H.M., Mohamed, E.M. and Salah, M. (2019) Role of VEGF, CD105, and CD31 in the Prognosis of Colorectal Cancer Cases. Journal of Gastrointestinal Cancer, 50, 23-34.
https://doi.org/10.1007/s12029-017-0014-y

[54]   Siller-Matula, J.M., Schwameis, M., Blann, A., Mannhalter, C. and Jilma, B. (2011) Thrombin as a Multi-Functional Enzyme. Focus on in Vitro and in Vivo Effects. Thrombosis and Haemostasis, 106, 1020-1033.
https://doi.org/10.1160/TH10-11-0711

[55]   Darmoul, D., Gratio, V., Devaud, H., Peiretti, F. and Laburthe, M. (2004) Activation of Proteinase-Activated Receptor 1 Promotes Human Colon Cancer Cell Proliferation through Epidermal Growth Factor Receptor Transactivation. Molecular Cancer Research, 2, 514-522.
https://doi.org/10.1074/jbc.M401430200

[56]   Chang, L.H., Chen, C.H., Huang, D.Y., Pai, H.C., Pan, S.L. and Teng, C.M. (2011) Thrombin Induces Expression of Twist and Cell Motility via the Hypoxia-Inducible Factor-1α Translational Pathway in Colorectal Cancer Cells. Journal of Cellular Physiology, 226, 1060-1068.
https://doi.org/10.1002/jcp.22428

[57]   Sangole, P.N. and Majumdar, A.S. (2018) Therapeutic Promise of Dabigatran Etexilate, an Oral Direct Thrombin Inhibitor in a Preclinical Model of Colon Carcinogenesis. Journal of Clinical and Experimental Pharmacology, 8, 247.

 
 
Top