Binding of unmodified and O-sulfated heparins to PF4.
After a stable baseline tracing was established by perfusion of unsupplemented buffer over biotinylated PF4 immobilized on a streptavidin chip in a BIAcore® biosensor equipment, a range of concentrations of unmodified heparin (HEP) or O-sulfated heparin (O-S HEP) were perfused over the chip. Binding of each GAG was measured in resonance units (1 RU = 1 pg/mm2) in triplicate at each concentration at 5 different concentrations. Representative binding curves are shown for the following concentrations of GAGs: 20.8 μmol/L HEP and 31.3 μmol/L O-S HEP. The perfusion of GAGs resulted in a shift in the tracing due to binding of the GAGs to PF4 (association phase; shaded area labeled A). When GAG perfusion was stopped and perfusion with unsupplemented buffer resumed, the tracing returned downward as the bound GAGs dissociated from PF4 (dissociation phase; shaded area labeled D). Perfusion of heparin resulted in a sharp up-slope, indicating rapid association, and a sharp down-slope following cessation of heparin perfusion, indicating rapid dissociation. In contrast, perfusion of O-sulfated heparin resulted in more gradual up-slope and down-slope, indicating slower association and dissociation from PF4. The table shows the median values for KD, ka (association rate) and kd(dissociation rate).
