Preparation of Fibrin Monomers from Human Fibrinogen in Urea, Using Soluble or Insolubilized Thrombin

Abstract

Fibrin Monomers were prepared by exposing human fibrinogen to thrombin in the presence of 3.3 m urea, pH 7.4 and +20 °C. Both with thrombin covalently bound to Sepharose 4 B and with thrombin in solution (100 NIH units/ml), extensive conversion of fibrinogen to fibrin within 1 h was achieved. No spontaneous clotting occurred.The fibrin preparations consisted to a very large extent of fibrin monomers, as evidenced by N-terminal analysis, agarose gel chromatography and SDS-polyacrylamide gel electrophoresis. When the urea concentration was reduced by dilution, the fibrin monomers retained their normal clottability, polymerization speed and cross-linking ability. Identical results were obtained with radioactive fibrin monomers prepared from ¹²⁵I-labelled fibrinogen. Storage for 4 weeks at +4 °C or quick freezing in liquid nitrogen and subsequent thawing caused only slightly prolonged polymerization times. Attempts to separate thrombin in solution from fibrin monomers by chromatography were unsuccessful, whereas this was easily achieved with insolubilized thrombin. Since traces of thrombin may influence the experimental results, the latter technique should be preferred.