The interaction of platelet factor four and glycosaminoglycans

Abstract

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 X a. 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 (< M r〉) = 11,000) was specific and saturable, with a molar stoichiometry of PF-4 to heparin of approximately 4:1 and an apparent estimated K d of 3 × 10 −8 m. Heparin fractions (< M r〉) = 6,000) with either low or high affinity for antithrombin III bound to PF-4 with a similar apparent K d . PF-4 also bound to dextran sulfate (< M r〉 = 22,500) with an estimated apparent K d of 6 × 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 IC 50 for heparin binding shifted from 10 to 150 n m. These studies demonstrate 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.