A Microcantilever Device to Assess the Effect of Force on the Lifetime of Selectin-Carbohydrate Bonds

摘要

A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-μm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewisx, was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), rf. From the distribution of forces at failure, the average force was determined and plotted as a function of ln rf. The slope and intercept of the plot yield the unstressed reverse reaction rate, kro , and a parameter that describes the force dependence of reverse reaction rates, ro. The ligand was titrated so adhesion occurred in ~30% of tests, implying that \u3e80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are ro = 0.048 and 0.016 nm and kro = 0.72 and 2.2 s-1 for loading rates in the ranges 200–1000 and 1000–5000 pN s-1, respectively. Levenberg-Marquardt fitting across all values of rf gives ro = 0.034 nm and kro = 0.82 s-1. The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations.