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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)
EL Yeo, JA Sheppard and IA Feuerstein
Department of Medicine, Toronto Hospital, University of Toronto, Canada.
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.
Volume 83,
Issue 9,
pp. 2498-2507,
05/01/1994
Copyright © 1994 by The American Society of Hematology
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