Purification and binding properties of human platelet factor four.
Methods are described for the purification of a heparin-neutralizing protein from human platelets. The protein, obtained by conventional or affinity chromatographic techniques, is homogeneous by disc and sodium dodecyl sulfate gel electrophoresis, immunoelectrophoresis, and gel electrofocusing and can be obtained in a final yield of 75%. The protein has a subunit molecular weight of 9600, an isoelectric point at pH 7.6, and 18% basic and 22% acidic amino acid residues. The purified heparin-neutralizing protein forms dissociable complexes with heparin as measured by electrophoretic and Millipore filtration techniques employing [3H]heparin. The ability of a series of sulfated glycosaminoglycans to displace [3H]heparin from the binding protein was compared. The mole ratios required were: heparin less than heparan sulfate less than dermatan sulfate less than chondroitin 6-sulfate less than chondroitin 4-sulfate. Although the degree of sulfation of the aminoglycans correlated with the ability to displace [3H]heparin, the conformation fo the carboxyl group of the uronic acid and the location of the sulfate groups on the amino sugar also influenced the affinity for the protein. Evidence is also presented that binding to aminoglycans occurs via ionic interactions between lysine residues on the protein and negatively charged groups on the aminoglycan. Chemical modification of lysines by guanidination decreased heparin-neutralizing and binding activity, while modification of arginine residues had no effect. Heparin could prevent lysine modification when specifically bound to the heparin-neutralizing protein, but did not prevent lysine modification of other proteins.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001779:Blood Coagulation Factors / Q000302:isolation & purification; D004553:Electric Conductivity; D006025:Glycosaminoglycans; D006493:Heparin; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D046911:Macromolecular Substances; D010978:Platelet Factor 4 / Q000302:isolation & purification; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D001779:Blood Coagulation Factors; D006025:Glycosaminoglycans; D046911:Macromolecular Substances; D010978:Platelet Factor 4; D006493:Heparin