Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin

P-selectin (GMP-140, PADGEM, CD62P) is a cell adhesion receptor which is believed to play an important role in inflammatory diseases by supporting leucocyte rolling. P-selectin is located on the granule membrane of Weibel-Palade bodies in resting endothelial cells and is expressed on the cell surface during cellular activation with various stimulators such as thrombin. Thereafter, P-selectin is internalized and sorted to the Golgi region and Weibel-Palade bodies again. However, whether P-selectin is re-expressed upon subsequent cellular stimulation has, to date, been unclear. To address this question, we measured the cellular content and surface expression of P-selectin, using indirect immunofluorescence and confocal laser cytometry. Surface expression of P-selectin reached a maximum <2 min after thrombin stimulation and declined to basal levels after 180 min. Rechallenge with thrombin induced rapid surface re-expression of P-selectin, which was independent of de novo protein synthesis, since cycloheximide did not inhibit re-expression. Moreover, re-expressed P-selectin supported the adherence of HL60 promyelocytic cells. These results clearly demonstrated that functional P-selectin molecule was recycled after repeated stimulation with thrombin, raising the possibility that P-selectin is involved in chronic inflammation.