Mode of interaction between platelet factor 4 and heparin.
Platelet factor 4 (PF4) is a platelet-derived protein capable of binding to, and thus neutralizing, the biological activities of heparin and heparan sulphate. The mode of binding of PF4 to heparin was investigated in a comparative study also involving antithrombin (AT; previously shown to selectively bind a specific oligosaccharide sequence) and fibronectin (FN; non-specific electrostatic interaction). Heparin-derived saccharides were incubated with each of the three proteins, followed by separation of free and protein-bound carbohydrate on a nitrocellulose filter. The interaction systems involved either (i) competition for the protein ligand between 3H-labelled heparin and unlabelled, size-fractionated heparin oligosaccharides (isolated after deaminative cleavage with HNO2) or (ii) direct binding of 3H-labelled oligosaccharides. Species smaller than octasaccharides were unable to bind AT, whereas binding to FN and PF4 increased continuously throughout the series, with increasing size of the oligosaccharides. Further separation by anion-exchange chromatography showed that the PF4-binding and FN-binding octasaccharides represented essentially all components present in the initial octasaccharide fraction, the proportion of binding species increasing with charge (hence with the degree of sulphation). The AT-binding octasaccharides, on the other hand, selectively represented only a few of the total octasaccharide components, without any correlation to overall charge. These results indicate that the binding of PF4 to heparin occurs by relatively non-specific electrostatic interactions. The methodology delineated here may be generally useful in assessing specificity in glycosaminoglycan-protein interactions.
Maccarana
M
M
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Lindahl
U
U
D000818:Animals; D000991:Antithrombins / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002417:Cattle; D002852:Chromatography, Ion Exchange; D005353:Fibronectins / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D010978:Platelet Factor 4 / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013464:Sulfuric Acids / Q000032:analysis; D013552:Swine; D014316:Tritium
D000991:Antithrombins; D005353:Fibronectins; D009844:Oligosaccharides; D013464:Sulfuric Acids; D014316:Tritium; D010978:Platelet Factor 4; D006493:Heparin