P-selectin glycoprotein ligand-l (PSGL-1) is a sialomucin molecule on leukocyte surface that interacts with all three selectins. In inflammatory and thrombotic processes, flowing leukocytes use PSGL-1 to tether to and roll on P-selectin on activated platelets and endothelial cells and on L-selectin on adherent leukocytes. Critical parameters to this function are kinetic rates and their force- and distance-dependence. This is due to the requirement for capture of rapidly moving cells from flow during brief encounters in a mechanically stressful environment. Extending the NIH grant (R01 AI044902, """"""""Kinetic Properties of Selectins""""""""), which focuses on the structure-function analysis using selectin mutants by the micropipette technique, we propose to use the atomic force microscope (AFM) to measure and compare the kinetic properties of selectin/PSGL-1 interactions using PSGL-1 mutants. Our central hypothesis is that the structure of PSGL-1 determines the PSGL-1 function via its effects on the kinetic rates of its interactions with selectins. This hypothesis will be tested by studies complementary to the parent grant, which are organized in two specific aims: 1. to determine the effects of structural variations in PSGL-1 on the kinetic rates and affinity of its interaction with three selectins; and 2. to examine the influences of lateral distance, approaching speed, and contact force on the forward rate of P-selectin/PSGL-1 interaction. The results of this project promise to improve our understanding of the biophysical basis of the selectin/PSGL-1 interaction-mediated cell adhesion at the molecular level. It may also provide guidance to the development of PSGL-1 and/or selectin-based drugs for treatment of inflammatory and thrombotic disorders.
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