Fibrinogen, 3×10-5 M, incubated at 25°C with thrombin 10-10 M, is slowly transformed to fibrin. Fibrinopeptide A (FPA) is released at a rate of approximately 1 nM/ml per min, and after 30 min a clot is formed. Prior to clot formation serially timed solution aliquots were examined by gel permeation chromatography on a Bio Gel 5M column (void volume 140 ml). High molecular weight complex concentration and molecular size increased with incubation time. Fibrinogen antigen and FPA concentration was determined for each effluent fraction and relative FPA content (ng FPA/μg fibrinogen equivalent) was linearly related to chromatographic effluent volume between 160 and 260 ml (Ve for fibrinogen 255 ml). Computer analysis of the effluent profiles indicated a linear fall in monomeric fibrinogen to 20% of original concentration, a linear increase in “fibrin” dimer (2 fibrinogen equivalents with 2 FPA) to 30% and in trimer (3 fibrinogen equivalents with 2 FPA) to 20% of original concentration with higher polymers accounting for the remaining protein. These complexes were stable and remained in solution after fractionation. Fibrin monomer, in 3 M urea and devoid of FPA, when added to fibrinogen resulted in clot formation, equivalent to 66% of added monomer, but minimal complex formation, 5% of total fibrinogen equivalents. Thus intermediate fibrin polymers which contain residual FPA are stable soluble molecular entities in contradistinction to fibrin monomer lacking FPA. Fibrin proteolysis products added to fibrinogen also show minimal complex formation with fibrinogen if the products are free of thrombin and FPA. Patient plasma samples can be similarly assayed and distinction can be made between high molecular weight complexes derived from fibrinogen through thrombin action or from fibrin through plasmin action.
Fucose content of keratan sulphates from bovine articular cartilage
