Granule-mediated release of sphingosine-1-phosphate by activated platelets

Sphingosine-1-phosphate (Si P) is an intracellularly generated bioactive lipid essential for development, vascular integrity, and immunity. These functions are mediated by SIP-selective cell surface G-protein coupled receptors. SIP signaling therefore requires extracellular release of this lipid. Several cell types release SIP and evidence for both plasma membrane transporter-mediated and vesicle-dependent secretion has been presented. Platelets are an important source of SIP and can release it in response to agonists generated at sites of vascular injury. SIP release from agonist-stimulated platelets was measured in the presence of a carrier molecule (albumin) using HPLC-MS/MS. The kinetics and agonist-dependence of SIP release were similar to that of other granule cargo e.g. platelet factor IV (PF4). Agonist-stimulated SIP release was defective in platelets from Unc13d(Jinx) (Munc13-4 null) mice demonstrating a critical role for regulated membrane fusion in this process. Consistent with this observation, platelets efficiently converted fluorescent NBD-sphingosine to its phosphorylated derivative Which accumulated in granules. Fractionation of platelet organelles revealed the presence of SIP in both the plasma membrane and in alpha-granules. Resting platelets contained a second pool of constitutively releasable SIP that was more rapidly labeled by exogenously added sphingosine. Our studies indicate that platelets contain two pools of SIP that are released extracellularly: a readily-exchangeable, metabolically active pool of S1P, perhaps in the plasma membrane, and a granular pool that requires platelet activation and regulated exocytosis for release. (C) 2014 Elsevier B.V. All rights reserved.