Sphingosine 1-phosphate formation and intracellular Ca2+ mobilization in human platelets: Evaluation with sphingosine kinase inhibitors

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cell ular signaling, acting intercellularly as well as intracellularly, In this study, we examined the role of Sph-1-P as a signaling molecule in human pla telets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C, Both DMS and DL-thre o-DHS were confirmed to be competitive inhibitors of Sph kinase obtained fr om platelet cytoplasmic fractions. In intact platelets labeled with [H-3]Sp h, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did no t affect [H-3]- Sph-1-P formation. While both DMS and DL-threo-DHS inhibite d not only [H-3]Sph-1-P formation but also protein kinase C-dependent plate let aggregation, staurosporine, apotent protein kinase inhibitor, only inhi bited the protein kinase C-dependent reaction. Hence, it is unlikely that S ph kinase activation and the resultant Sph-1-P formation are mediated by pr otein kinase C in platelets. Furthermore, Ca2+ mobilization induced by plat elet agonists that act on G protein-coupled receptor was not affected by DM S or DL-threo-DHS, Our results suggest that Sph-1-P does not mediate intrac ellular signaling, including Ca2+ mobilization, in platelets.