Immunovisualization of fibrinogen A alpha-chain heterogeneity in normal plasma and plasma from patients with DIC or on streptokinase therapy.
Purified fibrinogen as well as normal plasma, or plasma from patients with DIC or undergoing streptokinase(SK)-therapy was subjected to 1D- and 2D SDS-electrophoresis under reducing conditions. The pattern was revealed either by Coomassie-staining or immunostaining after Western-blotting and then compared. The use of polyclonal antibodies to fibrinogen as well as two monoclonal anti-bodies reacting with FPA and C-terminal part of the A alpha-chain confirmed immunologically the previously reported molecular weight heterogeneity of the A alpha-chain of the fibrinogen molecule as being a constituent of normal plasma, and lead to the following conclusions: 1. The MW-heterogeneity is observed in the fibrinogen pool of normal plasma as well as in DIC-plasma, SK-plasma and in purified fibrinogen, being the least noticeable in normal plasma and most advanced in SK-plasma. Patterns obtained using immunostaining with monoclonal anti-FPA confirm that the MW-heterogeneity of fibrinogen is mainly due to C-terminal degradation of the A alpha-chain. 2. Numerous A alpha-chain remnants (at least 9), with intact N-terminal ends, are found to be present in normal plasma, with a MW range from 66,200 to 36,000 Da, demonstrating that each of the "classical" HMW, LMW, LMW' subgroups consist of fibrinogen molecules which are very heterogeneous. 3. Two populations of A alpha-chains in purified fibrinogen and in fibrinogen in plasma react with the C-terminal specific Mab G-8. This is in contrast to the findings in plasma from streptokinase-treated patients, where several bands of lower molecular weights than the gamma-chain can be seen, suggesting the presence of free, circulating A-alpha chains split in the N-terminal half of the chain beyond the last inter-chain disulphide bond. 4. 2D electrophoresis disclosed substantial deviations in the patterns obtained with DIC-plasma, SK-plasma and with fibrinogen purified by beta-alanine-precipitation from that observed with normal plasma. The present technique allows selective characterization of fibrinogen independently of the other proteins present in plasma and offers extreme sensitivity.
Grøn
B
B
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Norway.
Bennick
A
A
Nieuwenhuizen
W
W
Bjørnsen
S
S
Brosstad
F
F
D015153:Blotting, Western; D004211:Disseminated Intravascular Coagulation / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000302:isolation & purification*; D005344:Fibrinopeptide A / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007120:Immunochemistry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D013300:Streptokinase / Q000494:pharmacology*
D010446:Peptide Fragments; D005344:Fibrinopeptide A; D005340:Fibrinogen; D013300:Streptokinase