DOWN-REGULATION OF GMP-140 (CD62 OR PADGEM) EXPRESSION ON PLATELETS BY N,N-DIMETHYL AND N,N,N-TRIMETHYL DERIVATIVES OF SPHINGOSINE

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with NN-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20-mu-M concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x)- or sialosyl-Le(x)-expressing cells with platelets and endothelial cells. They may therefore display effective inhibition of a variety of biological processes (e.g., inflammation and tumor metastasis) based on the expression and function of selectins.