Quantitative N-terminal analysis of fibrinogen-fibrin-related antigen [FR antigen] from human plasma

Abstract

Fibrinogen-fibrin-related antigen (FR antigen) was isolated from as little as 1 ml of human plasma by immuno-affinity chromatography with agarose-bound antibody to human fibrinogen. N-terminal analysis was performed to determine the nature and extent of proteolytic degradation of the FR antigen in patients with disseminated intravascular coagulation and in normal subjects. Thrombin cleavage of the A- and B-peptides from fibrinogen in vitro was monitored by the appearance of N-terminal glycine, and an increase in glycine was shown in the FR antigen of patients with disseminated intravascular coagulation. As plasmin progressively degraded fibrinogen, increases in N-terminal alanine, aspartic acid and lysine were observed, corresponding to the known plasmin-cleavage points of fibrinogen; increases in these N-terminal amino acids were also found in the patients' FR antigen. Thrombin treatment in vitro was used to remove fibrinopeptide A (N-terminal alanine) from the samples and to reflect specifically the N-terminal alanine at the plasmin-cleavage point (Arg-42-Ala-43) of the B beta-chain on assay; this alanine was increased progressively in the FR antigen of a patient during urokinase therapy, and was high in other patients when the FR antigen was examined by this procedure.

Isolation of Fibrinogen–Fibrin Related Antigen from Human Plasma by Immunoaffinity Chromatography: its Characterization in Normal Subjects and in Defibrinating Patients with Abruptio Placentae and Disseminated Cancer

“…The origin of much of the Ala remains uncertain. Recent results in our laboratory suggest that the normal control values offi chain Alanine are reduced by the use of a different column during high pressure liquid chromatography (Harris et al, 1979). However, in four of 18 patient samples the level was > 3 SD (99th percentile) above the normal value.…”

“…This was largely dependent on the amount of gamma globulin coupled to the agarose, e.g. there was more contamination when 3 6 mg of gamma globulin was coupled to each ml agarose than when 1.6 mg, and the least was found when the monospecific antibody was isolated, and 0.4 mg/ml was coupled to Sepharose (Harris et al, 1979). This was interpreted as protein-protein interaction rather than leakage from the column since negligible amounts of protein were eluted from control columns.…”

“…In this way, it was possible to avoid confusion with the normal N-terminal Ala of the Aa chain. Full details of these procedures are published elsewhere (Harris et al, 1979).…”