IMMUNO-VISUALIZATION OF FIBRINOGEN AND FIBRIN IN GELS PRDUCED BY GELATION OF PLASMA WITH ETHANOL

Abstract

The Ethanol Gelation Test(=EGT) is a well-documented simple,specific and frequently used test to detect plasma soluble fibrin(Godal & Abildgaard:Gelation of soluble fibrin in plasma by ethanol.Scand.J.Haanat.3,342,1966) .If soluble fibrin present in plasma amounts to 1% or more of the plasma-f ibrinogen conc.,the admixing of 0.15 ml 50% ethanol to 0.5 ml plasma in a test tube will-(subsequent to incubation for 10 min at 20°C)-upon tilting the test tube semi-horizontally produce a characteristic,(upwardly) convex gel. Although earlier studies have confirmed the validity and specificity of EGT as a means to detect soluble fibrin,we found it of interest to see to which degree such soluble fibrin is FXIII-stabilized EGT-positive(from patients with Disseminated Intravascular Coagula-tion(DIC) and EGT-negative plasma was studied as follows: The EGT test was performed as above,and the entire content of the test tube emptied upon a nylon micro-meshed membrane.Applying slight suction underneath the nylon manbrane the fluid was removed,leaving the ethanol precipitated material which was immediately dissolved in SDS (1%)-urea(5M)-Tris-HCl (0.15M,pH8.6). After incubation at 100°C for 1 min the material was SDS-electrophoresed on flat-bed agarose(2%) Subsequent to Western-blotting onto nitrocellulose and gelatine-blocking, the fibrin(ogen)-related pattern was reacted with either: a)polyclonal antibodies to fibrinogen,b)plyclonal antibodies to FPA or c)monoclonal antibody to FPA(gift from Dr.Nieuwenhuizen, Leyden, Holland) .Then,the fibrin(ogen)related pattern was developed using peroxidase-conjugated secondary antibodies.From the specificity of the primary antibodies used,it could be deduced that:1)A substantial amount of the soluble fibrin content of DIC-plasma was present in an oligomeric form(up to 6-mers) .2)These oligomers contained fibrinogen, i.e. thus representing FXIII-1 inked fibrinogen/fibrin hybride molecules .3) Even normal plasma contained some detectable oli-gomers(up to 3-mers) .4) Col lecting blood with all appropriate thrombin- and FXIII-inhibitors did not change the patterns obtained and described above.It may be concluded that soluble fibrin occurs mainly in a FXIII-stabilized,oligomeric form which contains fibrinogen Due to the nature of the polymerization process,the fibrinogen moiety of these hybride molecules must be end-located representing a physiological means to inhibit further polymer growth.