A new method for identification of adhesive interactions under shear force conditions

Adherence interactions under flow conditions are critical to a variety of hematologic processes. We have developed a novel adherence assay which examines, under defined shear conditions, interactions between cells or particles in flow and adhesion molecules bound to blotting membranes. Relevant molecules, either purified or as crude mixtures, can be applied directly to a membrane (e.g., dot/slot blot method) or bound following further analytic manipulations (e.g., PAGE and Western blot method). The membrane is then rendered transparent, incorporated into a parallel-plate flow chamber apparatus and mounted on an inverted microscope. Cells or particles bearing known adhesion molecules are introduced under controlled flow conditions and observed by video microscopy for interaction with the immobilized substrates; the relevant substrate molecules can also be immunostained for identification of particular components prior to assessment. In Western blots of cell extracts, rapid and reproducible identification of both novel and known adhesion molecules within the complex mixture can be achieved without requiring prior isolation or enrichment of the constituent parts. We have utilized this approach to examine the membrane structures on mature leukocytes and hematopoietic progenitor cells that mediate L-, P- and E-selectin binding. These studies verify that PSGL-1 on mature leukocytes serves as a ligand for all three selectins. However, on hematopoietic progenitors, in addition to PSGL-1, several other membrane glycoproteins (currently under study) serve as L-selectin ligands. Video recordings of these experiments will be presented to illustrate the utility of this new analytic technique.