The interaction of platelet factor four and glycosaminoglycans.
The interaction of platelet factor four (PF-4) with glycosaminoglycans (GAG) was evaluated using fluorescence spectroscopy, a radioligand binding assay, and a functional assay utilizing antithrombin III and factor Xa. In these studies, we have (i) characterized the binding parameters for PF-4 to several forms of heparin and to dextran sulfate; (ii) examined the structural features of these glycosaminoglycans which support PF-4 binding; and (iii) examined the effects of selective digestion of the carboxy terminus of PF-4 on binding. The binding of PF-4 to unfractionated porcine intestinal mucosal heparin ([Mr] = 11,000) was specific and saturable, with a molar stoichiometry of PF-4 to heparin of approximately 4:1 and an apparent estimated Kd of 3 X 10(-8) M. Heparin fractions ([Mr] = 6,000) with either low or high affinity for antithrombin III bound to PF-4 with a similar apparent Kd. PF-4 also bound to dextran sulfate ([Mr] = 22,500) with an estimated apparent Kd of 6 X 10(-8) M and a molar stoichiometry of approximately 16:1. Carboxypeptidase Y (CP-Y) digestion of PF-4 progressively decreased GAG binding. After 30 min of digestion, by which time all of the carboxyterminal serine and glutamate, both of the two leucines, and approximately one-quarter of the four lysines were removed, the IC50 for heparin binding shifted from 10 to 150 nM. These studies demonstrated the effect of GAG polymer size and degree of sulfation on the affinity and stoichiometry of PF-4 binding, and the critical importance of the carboxy-terminal amino acids of PF-4 for binding to natural and synthetic GAGs.
J
J
Loscalzo
B
B
Melnick
R I
RI
Handin
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001667:Binding, Competitive; D002268:Carboxypeptidases; D016264:Dextran Sulfate; D003911:Dextrans / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D006025:Glycosaminoglycans / Q000097:blood*; D006493:Heparin / Q000097:blood; D007413:Intestinal Mucosa / Q000378:metabolism; D010978:Platelet Factor 4 / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D013050:Spectrometry, Fluorescence; D013552:Swine
D003911:Dextrans; D006025:Glycosaminoglycans; D010978:Platelet Factor 4; D000990:Antithrombin III; D005170:Factor X; D006493:Heparin; D016264:Dextran Sulfate; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D015951:Factor Xa