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Partial Characterization of the Factor VIII‐Stimulating Material of Rabbit Liver Perfusates: Relationships to Rabbit Plasma Factor VIII
Author(s) -
Benson R. E.,
Jean Dodds W.
Publication year - 1975
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1975.tb00879.x
Subject(s) - sephadex , chemistry , chromatography , sepharose , antiserum , size exclusion chromatography , agarose , antibody , antigen , microbiology and biotechnology , polyacrylamide gel electrophoresis , lagomorpha , elution , sodium , biochemistry , enzyme , biology , endocrinology , immunology , organic chemistry
S ummary . Previous studies in our laboratory showed that effluent from perfused rabbit livers stimulates the production of factor‐VIII activity in perfused rabbit spleens. When concentrated supernatants of the hepatic perfusates were chromatographed on Sephadex G‐200, BioGel A‐15M and Sepharose 4B, the fractions with the greatest factor VIII‐stimulating capacity eluted in the void volume ( V °). Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the Sephadex G‐200 V ° fraction revealed components with a wide range of molecular weights. Rabbit factor VIII was isolated from ethanol‐concentrated, citrated rabbit plasma by Sepharose 4B filtration. The factor‐VIII activity appeared in the V ° when eluted with physiological saline but was retarded on columns eluted in 0.8 M sodium chloride. Further analysis with SDS gels demonstrated that the Sepharose‐purified factor‐VIII preparation entered the polyacrylamide gels only after reduction with 2‐mercaptoethanol. Goat antirabbit factor VIII antiserum prepared against the gel‐purified antigen contained factor VIII‐neutralizing activity, was apparently monospecific by double‐diffusion and produced faint, single peaks by electro‐immunodiffusion. Antibody neutralization studies with the antiserum showed that the concentrated supernatants of hepatic perfusates contain fourfold greater factor VIII‐neutralizing antigen than procoagulant activity, whereas control perfusates, lacking factor VIII‐stimulating capacity, had negligible antibody‐blocking effect. The presence in hepatic perfusates of both high‐molecular‐weight factor VIII‐stimulating material and large amounts of factor VIII‐neutralizing antigen lacking procoagulant activity suggests that they are interrelated.