Pselectin is the first biomolecule deployed from intracellular storage pools to the luminal surface. The interaction between Pselectin and its primary leukocyte-expressed ligand, P-selectin glycoprotein ligand-1, AbMole Clofentezine supports neutrophil rolling along the surface of the blood vessel. E-selectin, which binds to PSGL-1 among other ligands is also presented on the endothelium during the inflammatory response, but its expression is largely controlled by translation and its presentation temporally lags behind that of P-selectin in vivo. Given these temporal selectin expression dynamics, we felt it would be beneficial to independently probe bromelain’s effects on neutrophil interactions with substrates presenting each of these glycoproteins. To achieve this, we utilized a photochemical surface modification AbMole Metaproterenol Sulfate strategy developed in our lab to generate substrates 
presenting controlled densities of P-selectin or E-selectin, and then used these substrates to investigate the effect of bromelain treatment on the ability of human neutrophils to tether and roll in flow assays. Neutrophils are among the first responders that rapidly accumulate at sites of inflammation and thus a potential key player in the initial steps of the immune response. Flow cytometric expression analysis of two ligands involved in leukocyte recruitment mediated by P-selectin and E-selectin, PSGL-1 and cutaneous lymphocyte antigen, respectively, was performed to determine the effects of bromelain treatment on ligand expression. The results reveal a site-specific proteolysis through which bromelain treatment abolishes interactions between neutrophils and immobilized P-selectin, but not E-selectin, under conditions of physiological shear stress in vitro, suggesting another molecular mechanism through which bromelain may act as an anti-inflammatory agent. Interestingly, we found that bromelain treatment of neutrophils nearly eliminated their ability to interact with P-selectin presented on substrates in vitro, while E-selectin-mediated interactions are unaffected. The observations from neutrophil flow assays were complemented by dose-dependent effects of bromelain treatment on the expression of P-selectin and E-selectin ligands using flow cytometry. Our findings suggest that the anti-inflammatory effects of bromelain may be attributed in part to its ability to proteolytically process PSGL-1 and thereby reduce the number of cells interacting with P-selectin presented on the inflamed endothelium during the initial phases of an inflammatory response. Using a molecularly general method previously developed in our lab for the covalent and controllable photochemical immobilization of biomolecules on planar glass substrates, we generated and characterized substrates presenting defined “high” and “low” levels of P- or E-selectin.