Objective: The aim of the present study was to assess the antithrombotic properties of different strains of orally available antithrombotic lactic acid bacteria (LAB). Research Methods & Procedures: Antithrombotic activity, antiplatelet reactivity and/or thrombolytic activity, were measured in seven strains of LAB and LAB cell-free-extracts (LAB filtrates) using the shear-induced platelet reactivity/thrombolytic activity, The Global Thrombosis Test (GTT), with non-anticoagulated rat blood in vitro . Subsequently, the most potent antithrombotic strains identified in vitro we re assessed in vivo after oral administration in mouse carotid arteries using a helium-neon laser -induced thrombosis model. Result: Five strains out of seven LAB ( Lactobacillus paracasei KW 3100, Lactobacillus fermentum NBRC 3961, Lactobacillus pentosus JCM 8333, Leuconostoc oeni Elios 1, Pediococcus pentosaceus NK-2) promoted significant endogenous thrombolysis in vitro . In addition, one strain ( Lactobacillus fermentum NBRC 3961) significantly inhibited shear-induced platelet re- activity. Three antithrombotic strains, Lactobacillus fermentum NBRC 3961, Leuconostoc oeni Elios 1, and Lactobacillus pentosus JCM 8333, were further assessed in vivo . The results demonstrated that filtrates, and the cells per se of these LAB, modulated antiplatelet activity and/or thrombolytic a ctivity, and that the antithrombotic mechanisms were mainly influenced by protein content (60% - 70% of dry matter). Conclusion: The findings suggested that some strains of lactic acid bacteria could usefully provide the basis for the production of oral antithrombotic probiotics.
KeywordsAntithrombotic Lactic Acid BacteriaShear-Induced ThrombosisGlobal Thrombosis Test (GTT)Laser-Induced ThrombosisEndogenous Thrombolysis
Lichtenstein, A.H., Appel, L.J., Brands, M., Carnethon, M., Daniels, S., Franch, H.A., Franklin, B., Kris-Etherton, P., Harris, W.S., Howard, B., Karanja, N., Lefevre, M., Rudel, L., Sacks, F., Van Horn, L., Winston, M. and Wylie-Rosett, J. (2006) Summary of American Heart Association Diet and Lifestyle Recommendations Revision 2006. Arteriosclerosis, Thrombosis, and Vascular Biology, 26, 2186-2191. http://dx.doi.org/10.1161/01.ATV.0000238352.25222.5e
Masood, M.I., Qadir, M.I., Shirazi, J.H. and Khan, I.U. (2011) Beneficial Effects of Lactic Acid Bacteria on Human Beings. Critical Reviews in Microbiology, 37, 91-98. http://dx.doi.org/10.3109/1040841X.2010.536522
White, R.H. and Keenan, C.R. (2009) Effects of Race and Ethnicity on the Incidence of Venous Thromboembolism. Thrombosis Research, 123, S11-S17. http://dx.doi.org/10.1016/S0049-3848(09)70136-7
Rhee, S.J., Lee, J.-E. and Lee, C.-H. (2011) Importance of Lactic Acid Bacteria in Asian Fermented Foods. Microbial Cell Factories, 10, S5. http://dx.doi.org/10.1186/1475-2859-10-S1-S5
Tholstrup, T. (2006) Dairy Products and Cardiovascular Disease. Current Opinion in Lipidology, 17, 1-10.
Haro, C., Villena, J., Zelaya, H., Alvarez, S. and Aguero, G. (2009) Lactobacillus casei Modulates the Inflammation-Coagulation Interaction in a Pneumococcal Pneumonia Experimental Model. Journal of Inflammation, 6, 28. http://dx.doi.org/10.1186/1476-9255-6-28
Candela, M., Biagi, E., Centanni, M., Turroni, S., Vici, M., Musiani, F., Vitali, B., Bergmann, S., Hammerschmidt, S. and Brigidi, P. (2009) Bifidobacterial Enolase, a Cell Surface Receptor for Human Plasminogen Involved in the Interaction with the Host. Microbiology, 155, 3294-3303. http://dx.doi.org/10.1099/mic.0.028795-0
Kovács, I.B., Tigyi-Sebes, A., Trombitás, K. and Gorog, P. (1975) Evans Blue: An Ideal Energy-Absorbing Material to Produce Intravascular Microinjury by HE-NE Gas Laser. Microvascular Research, 10, 107-124. http://dx.doi.org/10.1016/0026-2862(75)90025-4
Yamamoto, J., Iizumi, H., Hirota, R., Shimonaka, K., Nagamatsu, Y., Horie, N. and Morita, S. (1989) Effect of Physical Training on Thrombotic Tendency in Rats: Decrease in Thrombotic Tendency Measured by the He-Ne Laser-Induced Thrombus Formation Method. Haemostasis, 19, 260-265.
Yamamoto, J., Yamashita, T., Ikarugi, H., Taka, T., Hashimoto, M., Ishii, H., Watanabe, S. and Kovacs, I.B. (2003) Gorog Thrombosis Test: A Global in Vitro Test of Platelet Function and Thrombolysis. Blood Coagulation & Fibrinolysis, 14, 31-39. http://dx.doi.org/10.1097/00001721-200301000-00007
Yamamoto, J., Inoue, N., Otsui, K., Ishii, H. and Gorog, D.A. (2014) Global Thrombosis Test (GTT) Detects Major Determinants of Hemostasis Including Platelet Reactivity, Endogenous Fibrinolytic and Thrombin Generating Potential. Thrombosis Research, 133, 919-926. http://dx.doi.org/10.1016/j.thromres.2014.02.018
Saraf, S., Christopoulos, C., Salha, I.B., Stott, D.J. and Gorog, D.A. (2010) Impaired Endogenous Thrombolysis in Acute Coronary Syndrome Patients Predicts Cardiovascular Death and Nonfatal Myocardial Infarction. Journal of the American College of Cardiology, 55, 2107-2115. http://dx.doi.org/10.1016/j.jacc.2010.01.033
Gorog, D.A., Yamamoto, J., Saraf, S., Ishii, H., Ijiri, Y., Ikarugi, H., Wellsted, D.M., Mori, M. and Yamori, Y. (2011) First Direct Comparison of Platelet Reactivity and Thrombolytic Status between Japanese and Western Volunteers: Possible Relationship to the “Japanese Paradox”. International Journal of Cardiology, 152, 43-48. http://dx.doi.org/10.1016/j.ijcard.2010.07.002
Yamamoto, J., Naemura, A., Ura, M., Ijiri, Y., Yamashita, T., Kurioka, A. and Koyama, A. (2006) Testing Various Fruits for Anti-Thrombotic Effect: I. Mulberries. Platelets, 17, 555-564. http://dx.doi.org/10.1080/09537100600759295
Yamamoto, J., Naemura, A., Ijiri, Y., Ogawa, K., Suzuki, T., Shimada, Y. and Giddings, J.C. (2008) The Antithrombotic Effects of Carrot Filtrates in Rats and Mice. Blood Coagulation & Fibrinolysis, 19, 785-792. http://dx.doi.org/10.1097/MBC.0b013e3283177b19
Fujiwara, D., Inoue, S., Wakabayashi, H. and Fujii, T. (2004) The Anti-Allergic Effects of Lactic Acid Bacteria Are Strain Dependent and Mediated by Effects on Both Th1/Th2 Cytokine Expression and Balance. International Archives of Allergy and Immunology, 135, 205-215. http://dx.doi.org/10.1159/000081305
Yamamoto, J., Taka, T., Yamada, K., Ijiri, Y., Murakami, M., Hirata, Y., Naemura, A., Hashimoto, M., Yamashita, T., Oiwa, K., Seki, J., Suganuma, H., Inakuma, T. and Yoshida, T. (2003) Tomatoes Have Natural Anti-Thrombotic Effects. British Journal of Nutrition, 90, 1031-1038. http://dx.doi.org/10.1079/BJN2003994
Shimizu, M., Sawashita, N., Morimatsu, F., Ichikawa, J., Taguchi, Y., Ijiri, Y. and Yamamoto, J. (2009) Antithrombotic Papain-Hydrolyzed Peptides Isolated from Pork Meat. Thrombosis Research, 123, 753-757. http://dx.doi.org/10.1016/j.thromres.2008.07.005
Ijiri, Y., Miura, M., Hashimoto, M., Fukunaga, C., Watanabe, S., Kubota, A., Oiwa, K., Okuda, T. and Yamamoto, J. (2002) A New Model to Evaluate the Diet-Induced Prothrombotic State, Using He-Ne Laser-Induced Thrombogenesis in the Carotid Artery of Apolipoprotein E-Deficient and Low-Density Lipoprotein Receptor-Deficient Mice. Blood Coagulation & Fibrinolysis, 13, 497-504. http://dx.doi.org/10.1097/00001721-200209000-00004
Hodge, J.E. and Hofreiter, B.T. (1962) Determination of Reducing Sugars and Carbohydrates. In: Whistler, R.L. and Wolfrom, M.L., Eds., Methods in Carbohydrate Chemistry, Vol. 1, Academic Press, New York, 380-394.
Gonnet, F., Lemaitre, G., Waksman, G. and Tortajada, J. (2003) MALDI/MS Peptide Mass Fingerprinting for Proteome Analysis: Identification of Hydrophobic Proteins Attached to Eucaryote Keratinocyte Cytoplasmic Membrane Using Different Matrices in Concert. Proteome Science, 1, 2. http://dx.doi.org/10.1186/1477-5956-1-2
Thiede, B., Hohenwarter, W., Alexander Krah, A., Mattow, J., Schmid, M., Schmidt, F. and Jungblut, P.R. (2005) Peptide Mass Fingerprinting. Methods, 35, 237-247. http://dx.doi.org/10.1016/j.ymeth.2004.08.015
Naemura, A., Mitani, T., Ijiri, Y., Tamura, Y., Yamashita, T. and Yamamoto, J. (2005) Anti-Thrombotic Effect of Strawberries. Blood Coagulation & Fibrinolysis, 16, 501-509. http://dx.doi.org/10.1097/01.mbc.0000184737.50594.a8
Yamamoto, J., Yamada, K., Naemura, A., Yamashita, T. and Arai, R. (2005) Testing Various Herbs for Antithrombotic Effect. Nutrition, 21, 580-587. http://dx.doi.org/10.1016/j.nut.2004.09.016
Naemura, A., Ijiri, Y., Kodama, M., Honda, Y. and Yamamoto, J. (2007-2008) A Rice Variety (HYJA-Ri4) Enhances Arterial Thrombus Formation in an Animal Thrombosis Model. Patho-physiology of Haemostasis and Thrombosis, 36, 241-244. http://dx.doi.org/10.1159/000252819
Ichiura, D., Naemura, A., Ura, M., Mori, M. and Yamamoto, J. (2009) Anti-Thrombotic Effect of Potato in Animal Experiments. In: Food, Vol. 3, Special Issue 2, Global Science Books, 8-12.
Hyodo, K., Horii, I., Nishino, M., Giddings, J.C. and Yamamoto, J. (2011) The Antithrombotic Effects of Onion Filtrates in Rats and Mice. Health, 3, 319-325. http://dx.doi.org/10.4236/health.2011.36055
Yamamoto, J., Masuda, M., Hyodo, K. and Iwasaki, M. (2012) Experimental Antithrombotic Effect of Potatoes Harvested in the Autumn. Health, 4, 108-112. http://dx.doi.org/10.4236/health.2012.42017
Morishita, M., Naemura, A., Tamura, Y., Yamaya, H., Tsuda, Y., Okada, Y., Okada, K., Matsuo, O. and Yamamoto, J. (2012) Mechanism of the Experimental Antithrombotic Effect of Some Apple Varieties Involves Enhanced Endogenous Thrombolytic Activity. Interventional Medicine & Applied Science, 4, 115-124.
Yamada, K., Naemura, A., Sawashita, N., Noguchi, Y. and Yamamoto, J. (2004) An Onion Variety Has Natural Antithrombotic Effect as Assessed by Thrombosis/Thrombolysis Models in Rodents. Thrombosis Research, 114, 213-220. http://dx.doi.org/10.1016/j.thromres.2004.06.007
Naemura, A., Ohira, H., Ikeda, M., Koshikawa, K., Ishii, H. and Yamamoto, J. (2006) An Experimentally Antithrombotic Strawberry Variety Is Also Effective in Humans. Pathophysiology of Haemostasis and Thrombosis, 35, 398-404. http://dx.doi.org/10.1159/000097696
Nishida, S., Michinaka, A., Nakashima, K., Iino, H. and Fujii, T. (2008) Evaluation of the Probiotic Potential of Lactobacillus paracasei KW3110 Based on in Vitro Tests and Oral Administration Tests in Healthy Adults. Journal of General and Applied Microbiology, 54, 267-276. http://dx.doi.org/10.2323/jgam.54.267
Boekhorst, J., Wels, M., Kleerebezem, M. and Siezen, R.J. (2006) The Predicted Secretome of Lactobacillus plantarum WCFS1 Sheds Light on Interactions with Its Environment. Microbiology, 152, 3175-3183. http://dx.doi.org/10.1099/mic.0.29217-0
Parkkinen, J. and Korhonen, T.K. (1989) Binding of Plasminogen to Escerichia coli Adhesion Protein. FEBS Letters, 250, 437-440. http://dx.doi.org/10.1016/0014-5793(89)80772-0
Schaumburg, J., Diekmann, O., Hagendorff, P., Bergmann, S., Rohde, M., Hammerschmidt, S., Jansch, L., Wehland, J. and Karst, U. (2004) The Cell Wall Subproteome of Listeria mono-cytogenes. Proteomics, 4, 2991-3006. http://dx.doi.org/10.1002/pmic.200400928
Lahteenmaki, K., Edelman, S. and Korthonen, T.K. (2005) Bacterial Metastasis: The Host Plasminogen System in Bacterial Invasion. Trends in Microbiology, 13, 79-85. http://dx.doi.org/10.1016/j.tim.2004.12.003
Sijbrandi, R., Den Blaauwen, T., Tame, J.R.H., Oudega, B., Luirink, J. and Otto, B.R. (2005) Characterization of an Iron-Regulated Alpha-Enolase of Bacteroides fragilis. Microbes and Infection, 7, 9-18. http://dx.doi.org/10.1016/j.micinf.2004.09.013
Bergmann, S. and Hammerschmidt, S. (2007) Fibrinolysis and Host Response in Bacterial Infections. Thrombosis and Haemostasis, 98, 512-520.
Hurmalainen, V., Edelman, S., Antikainen, J., Baumann, M., Lahteenmaki, K. and Korhonen, T.K. (2007) Extracellular Proteins of Lactobacillus crispatus Enhance Activation of Human Plasminogen. Microbiology, 153, 1112-1122. http://dx.doi.org/10.1099/mic.0.2006/000901-0
Candela, M., Miccoli, G., Bergmann, S., Turroni, S., Vitali, B., Hammerschmidt, S. and Brigidi, P. (2008) Plasminogen-Dependent Proteolytic Activity in Bifidobacterium lactis. Microbiology, 154, 2457-2462. http://dx.doi.org/10.1099/mic.0.2008/017095-0
Vastano, V., Capri, U., Candela, M., Sicilliano, R.A., Russo, L., Renda, M. and Sacco, M. (2013) Identification of Binding Sites of Lactobacillus plantarum Enolase Involved in the Interaction with Human Plasminogen. Microbiological Research, 168, 65-72. http://dx.doi.org/10.1016/j.micres.2012.10.001