title: Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model).
abstract: Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.
authors.affiliation: Department of Medicine, Toronto Hospital, University of Toronto, Canada.
authors.forename: E L
authors.initials: EL
authors.lastname: Yeo
authors.forename: J A
authors.initials: JA
authors.lastname: Sheppard
authors.forename: I A
authors.initials: IA
authors.lastname: Feuerstein
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001696:Biomechanical Phenomena; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000502:physiology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007962:Leukocytes / Q000502:physiology*; D007983:Lewis Blood Group Antigens / Q000276:immunology; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D019007:P-Selectin; D015539:Platelet Activation; D010973:Platelet Adhesiveness / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000502:physiology*; D019810:Sodium Azide
chemical_list: D000911:Antibodies, Monoclonal; D001386:Azides; D007983:Lewis Blood Group Antigens; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D019810:Sodium Azide; D008274:Magnesium; D002118:Calcium