Cellular deformation modulates leukocyte rolling on P-selectin and the formation of platelet thrombi on endothelium in shear

The selectin family of adhesion molecules plays an important role in leukocyte recruitment to the vascular endothelium during an inflammatory response. The engagement of endothelial P-selectin with leukocyte P-selectin glycoprotein ligand (PSGL-1) is involved in the initial leukocyte tether and rolling events. Agonist stimulated platelets are also able to express P-selectin and form circulating platelet-leukocyte aggregates in vivo. In this study, PSGL-1 coated microspheres were utilized to investigate both the role of localized leukocyte deformation in modulating the kinetics of P-selectin/PSGL-1 mediated leukocyte rolling and the significance of leukocyte protrusion into the flow field from the endothelium in directing the capture of platelets.; In the first stage of the project, a cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micro-mechanical properties of the neutrophil's surface microvilli, elastic structures on which PSGL-1 is localized. Microvilli extension is hypothesized to contribute to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin coated substrates were compared in a parallel-plate flow chamber.; The second phase of the study looked at the possible role of leukocytes adherent to endothelium in recruiting platelets and forming thrombi. Platelets can form circulating aggregates with neutrophils or monocytes under shear conditions. To evaluate the impact of neutrophils or monocytes on thrombi formation during an inflammatory response, platelets were perfused at venular wall shear stresses ranging between 0.3--8 dyn/cm2 over monocytes or neutrophils adherent on human umbilical vein endothelial cell (HUVECs) monolayers. Platelet recruitment to the endothelium via adherent leukocytes was significantly greater than platelet adhesion to the endothelium alone.; The final phase of the study investigated the localized fluid disturbance around adherent leukocytes and its effect on platelet accumulation. Neutrophils on cytokine-stimulated HUVECs appeared more activated than monocytes based on visual observation of ruffles and lamellipodia. Computational modeling of the local hydrodynamics around a transmigrating leukocyte reveals an approximate 6-fold deficit in the amount of shear stress reduction compared to a fully protruding leukocyte that has just initiated contact with the surface. (Abstract shortened by UMI.)...