(Authors): Merck Research Laboratories, Department of Pharmacology
Flow cytometry has been shown to be a useful tool in ligand/receptor interactions, calcium signaling, cell cycle analysis and kinetic measurements. We describe the design and the application of a flow cytometric cell that allows temperature control and addition of reagents to samples which are on-line in a flow cytometer. The cell was used in the flow cytometric measurements of dynamic and equilibrium binding parameters of RGD-peptidomimetic L-762,745, which is a potent fluorescien-containing inhibitor of platelet aggregration. The effect of temperature on the dynamic and equilibrium binding parameters of L-762,745 was measured using this custom-made chamber that was designed for a Becton/Dickinson FACScan or FACSCalibur flow cytometer. Kinetic binding measurements with this fluorescent ligand indicate a two-step binding mechanism which involves a conformational rearrangement of the receptor-ligand complex. The overall second order binding constants are several order of magnitude slower than for diffusion controlled processes. The values of k-1 and KD obtained by fitting the kinetic binding data in two-step model are in a good agreement with directly detected values of koff(L-762,745) = (1.9 ± 0.6) 10-3 s-1 and KD(L-762,745) = 12 ± 0.5 nM. The temperature dependence of dissociation of the fluorescent ligand L-762,745 from resting platelets showed a increase in values of koff with an increase in temperature (koff (17°C) = 1.49 ; koff (37°C) = 3.55 ). The values for enthalpy and entrophy are 32 kJ mol-1 and - 190JK-1mol-1 respectively.