ABB  Vol.2 No.1 , February 2011
Enzyme electrophoresis method in analysis of active components of haemostasis system
The novel modifications of substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis that can be used for the detection of proteases and its activators are reported. The protease/activator samples were separated on a protein substrate-SDS-polyacrylamide gel. To detect plasminogen activators fibrinogen and Glu-plasminogen were incorporated into the SDS-PAG followed by 1 h incubation at 37?C in thrombin solution (1 NIH/ml). After electrophoresis the gel was stained according to the standard protocol. To detect fibrin-unspecific plasminogen activators from snake venom incubation in thrombin solution was substituted for 12 h incubation in 50 mM Tris-HCl (pH 7.4). To detect fibrinogen-degrading enzymes fibrinogen-containing gel was used. Activity of protease/activator was visualized in the gel as clear bands against the dark background. These new techniques offer several advantages including determination of the quantity and activity of t-PA and urokinase, however cannot be recommended for precise quantification of activators; the total procedure is quite quick and simple; method is convenient tool for detection of novel protein-protein interactions in haemostasis system; the sensitivity of the method is ≤0.01 IU per track.

Cite this paper
nullOstapchenko, L. , Savchuk, O. and Burlova-Vasilieva, N. (2011) Enzyme electrophoresis method in analysis of active components of haemostasis system. Advances in Bioscience and Biotechnology, 2, 20-26. doi: 10.4236/abb.2011.21004.
[1]   Westermeeier, R. and Naven, T. (2002) Proteomics in practice: A laboratory manual of proteome analysis, Wiley-VCH Verlag GmbH.

[2]   Erickson, L., Lawrence, D. and Loskutoff, D. (1984) Reverse fibrin autography: A method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate—polyacrylamide gel electrophoresis. Analytical Biochemistry, 137, 454-463. doi:10.1016/0003-2697(84)90113-1

[3]   Brunner, G. and Schirrmacher, V. (1988) Fibrin autography of plasminogen activator by electrophoretic transfer into fibrin agar gels. Analytical Biochemistry, 168, 411-416. doi:10.1016/0003-2697(88)90337-5

[4]   Hanspal, J., Bushell, G. and Ghosh, P. (1983) Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical Biochemistry, 132, 288-293. doi:10.1016/0003-2697(83)90010-6

[5]   Zhao, Z. and Russell, P. (2003) Trypsin activity assay in substrate-specific one- and two-dimensional gels: A powerful method to separate and characterize novel proteases in active form in biological samples. Electrophoresis, 24, 3284-3288. doi:10.1002/elps.200305531

[6]   Le, Q., Ohashi, A., Hirose, S. and Katunuma, N. (2005) Reverse zymography using fluorogenic substrates for protease inhibitor detection. Electrophoresis, 26, 1038- 1045. doi:10.1002/elps.200306142

[7]   Wilkesman, J. and Schroder, H. (2007) Analysis of serine proteases from marine sponges by 2-D zymography. Electrophoresis, 28, 429-436. doi:10.1002/elps.200600332

[8]   Le, Q. and Katunuma, N. (2004) Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system. Analytical Biochemistry, 324, 237-240. doi:10.1016/j.ab.2003.09.033

[9]   Martinez, M., Newbold, C., Wallace, R. and Movano, F. (2002) Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electrophoresis, 23, 1-7.

[10]   Vazquez, D. and Peyronel, A.C. (1995) A simple and rapid technique for postelectrophoretic detection of proteases using azocasein. Electrophoresis, 16, 1894-1897.

[11]   Fareed, J., Hoppensteadt, D. and Leya, F. (1998) Useful laboratory tests for studying thrombogenesis in acute cardiac syndromes. Clinical Chemistry, 44, 1845-1853.

[12]   Zawilska, K. (1995) Progress in the detection of intravascular activation of fibrinolysis. Acta haematologica polonica, 26, 33-38.

[13]   Bu, C., Zhang, C. and Li, Z. (2007) Autoantibodies to plasminogen and tissue plasminogen activator in women with recurrent pregnancy loss. Clinical & Experimental Immunology, 149, 31-39. doi:10.1111/j.1365-2249.2007.03382.x

[14]   Hoylaerts, M., Rijken, D., Lijnen, H. and Collen, D. (1982) Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. Journal of Biological Chemistry, 257, 2912-2919.

[15]   Protein Electrophoresis. (1999) Technical manual, Amersham Biosciences Inc.

[16]   Heussen, C. and Dowdle, E. (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Analytical Biochemistry, 102, 196-202. doi:10.1016/0003-2697(80)90338-3

[17]   Harlow, E. and Lane, D. (1988) Antibodies. Cold Spring Harbor Laboratory, New York.

[18]   Chibber, B., Deutsch, D. and Mertz, E. (1974) Affinity chromatography of plasminogen. Methods in Enzymology, 34, 424-432.

[19]   Wiman, B. and Wallen, P. (1973) Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound. European Journal of Biochemistry, 36, 25-31. doi:10.1111/j.1432-1033.1973.tb02880.x

[20]   March, S., Parikh, I. and Cuatrecasas, P. (1974) A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Analytical Biochemistry, 60, 149-152. doi:10.1016/0003-2697(74)90139-0

[21]   Krasnobryzha, E., Savchuk, O. and Volkov, G. (2004) Study of streptokinase influence on the haemostasis system parameters in model systems in vivo. Ukrainian Biochemical Journal, 76, 56-61.

[22]   Savchuk, O., Levkin, M., Karbovskyy, V., Gornitskaya, O., Volkov, G. and Tseren, B. (2006) Plasminogen activatof from Agkistrodon halys halys venom. Ukrainian Biochemical Journal, 78, 32-37.

[23]   Platonova, T., Gornitskaya, O., Metelitsina, I. and Savchuk, O. (2001) Components of blood clotting system and fibrinolytic system in subretinal fluid at regmatogenic retina exfoliation. Medichna Chimia, 3, 5-8.

[24]   Petik, A., Platonova, T. and Savchuk, O. (2001) Indexes of haemostasis system during Lewis carcinoma growth. Experimental Oncology, 23, 73-75.

[25]   Zhukova, A., Krasnobrysha, I. Savchuk, O. and Volkov, G. (2009) The investigation of protein-protein interactions in haemostasis system using enzyme electrophoresis method. XXII Congress of the International Society of Thrombosis and Haemostasis. Journal of Thrombosis and Haemostasis, 7, Boston, USA, 367.

[26]   Bruhn, H., Conard, J., Mannucci, M., Monteagudo, J., Pelzer, H., Reverter, J., Samama, M., Tripodi, A. and Wagner, C. (1992) Multicentric evaluation of a new assay for prothrombin fragment F1 + 2 determination. Thromb Haemost, 68, 413-417.

[27]   Ferlito, S., Gallina, M., Mangiameli, S. and Chiaranda, G. (1995) Thrombotic markers during myocardial infarctionPanminerva. Panminerva Medica, 37, 133-136.

[28]   Granger, C., Becker, R., Tracy, R., Califf, R., Topol, E., Pieper, K., Ross, A., Roth, S., Lambrew, C. and Bovill, E. (1998) Thrombin generation, inhibition and clinical outcomes in patients with acute myocardial infarction treated with thrombolytic therapy and heparin: Results from the GUSTO-I Trial. GUSTO-I Hemostasis Substudy Group. Global Utilization of Streptokinase and TPA for Occluded Coronary Arteries. Journal of the American College of Cardiology, 31, 497-505. doi:10.1016/S0735-1097(97)00539-1

[29]   Johns, J., Gold, H., Leinbach, R., Yasuda, T., Gimple, L., Werner, W., Finkelstein, D., Newell, J., Ziskind, A. and Collen, D. (1988) Prevention of coronary artery reocclusion and reduction in late coronary artery stenosis after thrombolytic therapy in patients with acute myocardial infarction. A randomized study of maintenance infusion of recombinant human tissue-type plasminogen activator. Circulation, 78, 546-556.

[30]   Nilsen, D., Goransson, L., Larsen, A., Hetland, O. and Kierulf, P. (1997) Systemic thrombin generation and activity resistant to low molecular weight heparin administered prior to streptokinase in patients with acute myocardial infarction. Tromb Haemost, 77, 57-61.

[31]   Chandler, W. and Stratton, J. (1994) Laboratory evaluation of fibrinolysis in patients with a history of myocardial infarction. American Journal of Clinical Pathology, 102, 248-252.

[32]   Genser, N., Lechleitner, P., Maier, J., Dienstl, F., Artner-Dworzak, E., Puschendorf, B. and Mair, J. (1998) Rebound increase of plasminogen activator inhibitor type I after cessation of thrombolytic treatment for acute myocardial infarction is independent of type of plasminogen activator used. Clinical Chemistry, 44, 209-214.

[33]   Yamada, S., Yamada, R., Ishii, A., Ashikawa, K., Kawamitsu, H. and Fujita, K. (1996) Evaluation of tissue plasminogen activator and plasminogen activator inhibitor-I levels in acute myocardial infarction. Journal of Cardiology, 27, 171-178.

[34]   Markland, F. (1998) Snake venoms and the hemostatic system. Toxicon, 36, 1749-1800. doi:10.1016/S0041-0101(98)00126-3

[35]   Immonen, I., Konttinen, Y., Sorsa, T., Tommila, P. and Sirén, V. (1996) Proteinases in subretinal fluid. Graefes Archive for Clinical and Experimental Ophthalmology, 234, 105-109. doi:10.1007/BF00695249

[36]   O’Reilly, M. (1997) Angiostatin: An endogenous inhibitor of angiogenesis and of tumor growth. EXS, 79, 273-294.

[37]   Geiger, J. and Cnudde, S. (2004) What the structure of angiostatin may tell us about its mechanism of action. Thromb Haemost, 2, 23-34. doi:10.1111/j.1538-7836.2004.00544.x

[38]   Chen, Y., Wu, H., Li, C., Huang, Y., Chiang, C., Wu, M. and Wu, L. (2006) Anti-angiogenesis mediated by angiostatin K1-3, K1-4 and K1-4.5. Involvement of p53, FasL, AKT and mRNA deregulation. Thromb Haemost, 95, 668-677.

[39]   Wang, H., Doll, J., Jiang, K., Cundiff, D., Czarnecki, J., Wilson, M., Ridge, K. and Soff, G. (2006) Differential binding of plasminogen, plasmin, and angiostatin 4.5 to cell surface beta-actin: Implications for cancer-mediated angiogenesis. Cancer Research, 66, 7211-7215. doi:10.1158/0008-5472.CAN-05-4331

[40]   Kastrikina, T., Taran, L. and Kudinov, S. (1986) Kinetic characteristics of fibrinogen and fibrin hydrolysis by plasmin 1 and 2 and miniplasmin. Thromb Research, 41, 681-688. doi:10.1016/0049-3848(86)90365-8

[41]   Kolev, K., Komorowicz, E., Owen, W. and Machovich, R. (1996) Quantitative comparison of fibrin degradation with plasmin, miniplasmin, neurophil leukocyte elastase and cathepsin G. Thromb Haemost, 75, 140-146.

[42]   Kolev, K., Léránt, I., Tenekejiev, K. and Machovich, R. (1994) Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin, and miniplasmin by plasma protease inhibitors. Journal of Biological Chemistry, 269, 17030-17034.

[43]   Ponting, C., Marshall, J. and Cederholm-Williams, S. (1992) Plasminogen: A structural review. Blood Coagulation & Fibrinolysis, 3, 605-614.