Supports E-selectin recognition by ligands besides PSGL-1. Neutrophils flowed across simple human serum albuminblocked AbMole Folic acid substrates, without P- or E-selectin, showed no interactions which established that observed cell-substrate interactions were the result of specific receptor-ligand interactions. To further investigate the molecular mechanism by which AbMole Pamidronate disodium pentahydrate bromelain treatment regulates the initial phases of neutrophil tethering, we performed flow cytometric analyses to determine how expression of PSGL-1, which binds to both P-selectin and Eselectin, is modulated by bromelain treatment. There are two distinct structural domains of PSGL-1 that are required for interactions with each of the selectins and our flow cytometric analysis utilized two mAbs specific for these regions. Antibody KPL-1 recognizes a region that contains the sulfated tyrosine motif required for interactions with P-selectin, but not E-selectin. This specificity was previously demonstrated as incubation of leukocytes with the KPL-1 antibody completely blocked interactions with P-selectin without affecting leukocyte recognition of Eselectin. Antibody CHO131 recognizes a sialyl-Lewisx�C bearing a core 2 O-glycan that is required for interaction of neutrophils with both P-selectin and E-selectin. Flow cytometric analysis using these antibodies suggests that bromelain is able to specifically cleave PSGL-1 at a site in between the epitopes for clones KPL-1 and CHO131. However, analysis with clone CHO131 showed that the levels of this epitope were not significantly decreased at all concentrations of bromelain tested. In fact, CHO131 epitope expression moderately increased at low bromelain concentrations before showing a slight decrease at higher concentrations. While we do not completely understand the significance of this increase, it is clear that bromelain is able to directly and dramatically attenuate the sulfated tyrosine motif needed for P-selectin tethering while leaving significant expression of the sLex glycan needed for E-selectin’s interaction with PSGL-1. Treatment of human neutrophils with deactivated bromelain had no significant effect on neutrophil expression of PSGL-1, suggesting that enzyme activity is required to induce the changes in surface expression of PSGL-1 that we observed following treatment with active bromelain. The molecularlyspecific processing of PSGL-1 at a position that down-regulates interactions with P-selectin, yet upstream of the sialyl-Lewisx�C bearing core 2 O-glycan structure involved in E-selectin interactions, suggests that bromelain may selectively affect the very initial phases of neutrophil recruitment. As additional verification of PSGL-1 processing by bromelain, we performed Western blot analysis of the protein after exposure to the enzyme.