A Simple Rapid Method for Isolating Soluble Fibrin Complexes from Fibrinogen by Treatment with Thrombin and t-AMCHA

Summary

Using treatment with thrombin associated with trans-aminomethylcyclohexane carboxylic acid (t-AMCHA), a simple and rapid method for isolating soluble fibrin complexes (SFC) from fibrinogen in the plasma was developed. By this procedure, the recovery rates of SFC and early FDP (mainly X) increased according to the concentration of t-AMCHA, reaching a maximum at 286 mM t-AMCHA. On the other hand, the recovery rate of fibrinogen remained below 1.5% and that of late FDP was almost 100% at all concentrations of t- AMCHA. These results suggested that SFC and FDP could be isolated from fibrinogen by thrombin and t-AMCHA (286 mM) treatment. Moreover, it was possible to isolate SFC from FDP using gel filtration after treatment with thrombin and t-AMCHA. The SFC could be quantified by assay of the eluted fractions containing SFC by the staphylococcal clumping test.

• References

• 1 Fletcher AP, Alkjaersig NK. 1971; Blood hypercoagulability, intravascular coagulation and thrombosis. New diagnostic concepts Thromb. Diath. Haemorrh. 45 (Suppl.) 389
• 2 Hawiger J, Niewiarowski S, Gurewich V, Thomas DP. 1970; Measurement of fibrinogen and fibrin degradation products in serum by staphylococcal clumping test. J. Lab. Clin. Med. 75: 993
• 3 Hayashi S. 1979; Studies on the significance of serum soluble fibrin in nephritis, with special reference to the technique of staphylococcal clumping. Acta Haem. Jap. 42: 53
• 4 Heene DL, Matthias FR. 1973; Adsorption of fibrinogen derivatives on insolubilized fibrinogen and fibrin monomer. Thrombos. Res. 2: 137
• 5 Von Hugo R, Hafter R, Stemberger A, Graefe H. 1975; Soluble fibrin monomer complexes demonstrated by agarose gel filtration and by adsorption on insolubilized fibrinogen. Thromb. Diath. Haemorrh. 34: 216
• 6 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951; Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265
• 7 Matthias FR, Reinicke R, Heene DL. 1977; Affinity chromatography and quantification of soluble fibrin from plasma. Thrombos. Res. 10: 365
• 8 Weber K, Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406