GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood

Abstract

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally, but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer. Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47 degrees C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassie blue. Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.