Purification of a heparin-neutralizing protein from rabbit platelets and its homology with human platelet Factor 4.

Abstract

A heparin-neutralizing protein was purified in 50% yield from a rabbit platelet extract by heparin-sepharose affinity chromatography. The specific heparinneutralizing activity of this protein was 185 + 32 units/ mg. The purified protein gave a single Coomassie bluestaining band in sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of mercaptoethanol. In immunodiffusion analysis, an antibody raised against the purified material produced a single line of identity when reacted against the immunogen or against solubilized platelets. This rabbit platelet protein was compared with purified human platelet factor 4. Both proteins eluted from heparin-Sepharose at similar salt concentrations (1 to 1.5 M NaCl) and had similar heparin-neutralizing activities (rabbit = 185 units/mg, human = 225 units/ mg). A mixture of the two proteins co-sedimented into a 5 to 20% linear sucrose gradient. The 1311-labeled rabbit protein migrated as an apparently larger species when it was co-electrophoresed with lZ51-labeled human platelet factor 4 in sodium dodecyl sulfate polyacrylamide gels. This apparent size difference was corroborated by a corresponding difference in the minimum molecular weights calculated from amino acid compositions (rabbit = 8900, human = 7767). Nevertheless, the amino acid compositions of the two proteins were highly similar, both lacking phenylalanine, methionine, and tryptophan and having a minimum of 1 tyrosine per mole. The human platelet factor 4 bound to goat antibodies to rabbit protein and could displace the rabbit protein in a competitive radioimmunoassay for the rabbit protein. This indicated shared antigenic determinants between the two proteins. Finally, and most significantly, there was a high degree of NHz-terminal amino acid sequence identity when Cys residues of both proteins were aligned. Thus, by criteria including size, activity, amino acid composition, immunological crossreactivity, and partial amino acid sequence identity, the rabbit platelet heparin-neutralizing protein is homologous with human platelet factor 4. It is proposed that this protein be termed “rabbit platelet factor 4.”