Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

Summary

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.

The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.

It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Keywords

Thrombin - Meizothrombin - Meizothrombin-des fragment 1 - Transblotting enzymes

• References

• 1 Jackson CM, Nemerson Y. Blood coagulation. Annu Rev Biochem 1980; 49: 765-811
• 2 Rosing J, Tans G. Meizothrombin, a major product of factor Xa- catalyzed prothrombin activation. Thromb Haemostas 1988; 60: 355-360
• 3 Rosing J, Zwaal RF A, Tans G. Formation of meizothrombin as an intermediate in factor Xa-catalyzed prothrombin activation. J Biol Chem 1986; 261: 4224-4228
• 4 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem 1986; 261: 8977-8984
• 5 Boissel J-P, Le Bonniec B, Rabiet MJ, Labie D, Elion J. Covalent structures of β- and γ-autolytic derivatives of human α-thrombin. J Biol Chem 1984; 259: 5691-5697
• 6 Morita T, Iwanaga S, Suzuki T. The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venom and characterization of the new active intermedates. J Biochem 1976; 79: 1089-1108
• 7 Granelli-Pipemo A, Reich G. A study of proteases and protease- inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-234
• 8 Heussen G, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and co-polymerized substrates. Anal Biochem 1980; 102: 196-202
• 9 Wagner OF, Bergmann I, Binder BR. Chromogenic substrate autography: A method for the detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels. Anal Biochem 1985; 151: 7-12
• 10 van Rijn JL M L, Govers-Riemslag JW P, Zwaal RF A, Rosing J. Kinetic studies of prothrombin activation: Effect of factor Va and phospholipid on the formation of the enzyme-substrate complex. Biochemistry 1984; 21: 4557-4564
• 11 Tans G, Rosing J, Griffin JH. Sulfatide-dependent autoactivation of human blood coagulation factor XII (Hageman factor). J Biol Chem 1983; 258: 8215-8222
• 12 Laemmli UK. Cleavage of structural protein during the assembly of the head bacteriophage T4. Nature 1970; 227: 680-685
• 13 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4356
• 14 van der Graaf F, Tans G, Bouma BN, Griffin JH. Isolation and functional properties of the heavy and light chains of human plasma kallikrein. J Biol Chem 1982; 257: 14300-14305
• 15 Fujikawa K, Legaz ME, Davie EW. Bovine factor X_x and X₂ (Stuart factor). Isolation and characterization. Biochemistry 1972; 11: 4882-4891
• 16 Thaler E, Schmer G. A simple two-step isolation procedure for human and bovine antithrombin II/III (heparin cofactor): a comparison of two methods. Br J Haematol 1975; 31: 233-243
• 17 di Scipio RG, Hermodson HA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor) and protein S. Biochemistry 1977; 698-706
• 18 Dahlbäck B. Human coagulation factor V purification and thrombin- catalyzed activation. J Clin Invest 1980; 66: 583-591
• 19 Fujikawa K, Legaz ME, Davie EW. Bovine factor X_x (Stuart factor). Mechanism of activation by a protein from Russel’s viper venom. Biochemistry 1972; 11: 4892-4899
• 20 Schiffman S, Theodor J, Rapaport SJ. Separation from Russel’s viper venom of one fraction reacting with factor X and another reacting with factor V. Biochemistry 1969; 8: 1397-1405
• 21 Rhee M-J, Morris S, Kosow DP. Role of meizothrombin and meizothrombin (des F1) in the conversion of prothrombin to thrombin by the Echis carinatus venom coagulant. Biochemistry 1982; 21: 3437-3443
• 22 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 23 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 24 Robbins KC, Summaria L, Wohl RC. Human plasmin. Methods Enzymol 1981; 80: 379-387
• 25 Downing MR, Butkowski R, Clark MR, Mann KG. Human prothrombin activation. J Biol Chem 1975; 250: 8897-9069