Affinity and kinetics of selectin mediated leukocyte adhesion
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Cell adhesion molecules belonging to the selectin family have been shown to facilitate leukocyte trafficking to sites of inflammation and injury. Selectins have been shown to play an important role during the normal immune response, many inflammatory diseases and in several cardiovascular disorders. These processes involve selectins recognizing carbohydrate moieties displayed on their natural glycoprotein ligands under shear conditions in vivo. Therefore, we have focused on developing novel measurement and analysis techniques to quantify selectins binding to their naturally occurring ligands on neutrophils under physiologically relevant shear forces. These techniques could then be applied to quantify the efficacy of oligosaccharide antagonists or effects of structural modifications in selectin on binding properties. The initial work focuses on developing experimental techniques to quantify binding kinetics and affinity of selectins with their natural ligand on neutrophils under both static and shear conditions. We chose to focus on flow cytometry methods for numerous reasons. First, they require a relatively small volume which can help conserve reagents that scarcely available. Second, there are thousands of binding events or collisions which results in more reliability in data generated. Isolated human neutrophils were used in many assays in order to closely mimic the natural valency of selectins and their ligands, since this feature may affect selectin-ligand recognition specificity. Finally, the protocol did not require wash steps which is critical when you are quantifying binding events with rapid binding kinetics. Next, we tested the possibility that synthetic carbohydrates based on the exact structures of natural glycans expressed on PSGL-1 and GlyCAM-1 can be superior inhibitors of selectin binding. We utilized the methods developed earlier to screen various small molecules. Our studies suggest that the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. We identify a novel molecule Galβ1,4(Fucα1,3)GlcNAcβ1,6(GalNAcβ1,3)GalNAcα-OMe which blocked L- and P-selectin binding at 30-100 fold lower doses than sLeX. This molecule may represent a candidate for future drug development. Finally, in the first measurements of the affinity and kinetics of selectin interaction with carbohydrates, we observed that a sLeX mimetic (TBC1269) bound P-selectin with a dissociation constant ( K D ) of ∼111.4μM, and very rapid association and dissociation rates ( k off > 3/s and k on > 27,000/M/s). Finally, we investigated whether the molecular length of L-selectin results in the 'shear threshold' behavior observed previously. A domain swapping experiment between L- and P-selectin resulted in long and short selectin molecules with the binding specificity of either L- or P-selectin depending on the Lectin domain present. Lengthening L-selectin resulted in higher binding levels. Further experiments were performed to determine if the enhanced binding was due to the interactions of the selectin with the cellular glycocalyx by removing the glycocalyx components. Removal of both heparin sulfate and chondroitin sulfate did not alter the binding kinetics of the selectin constructs.