Abstract 16950: Sphingosine-1-Phosphate is Thromboxane-dependently Released from Platelets and Modulates Chemotaxis of Human Monocytes
Sphingosine-1-phosphate (S1P) is a sphingosine-derived lipid signaling molecule and an important mediator of vascular homeostasis. Upon vascular injury, S1P is released from platelets by thrombin. The underlying mechanisms and functional consequences are to date incompletely understood. Here, we describe that the release of S1P from human platelets induced after activation of the protease-activated receptor-1 (PAR-1) by thrombin dependent on thromboxane (TX) formation. Platelet-derived S1P modulates human monocyte migration which is attenuated after inhibition of TX formation. S1P was detected by mass spectrometry and by thin layer chromatography in [3H]sphingosine-labeled platelets. Human peripheral blood monocytes were isolated by ficoll gradient centrifugation and cleared from contaminating leukocytes by antibody-coated beads. Migration was determined in modified Boyden chamber assays. RNA interference was used to knock down S1P receptor expression. Release of S1P was stimulated about 5 fold (0.3±0.1 vs. 1.6±0.5 nmol/1x106 platelets) by the PAR-1-activating-peptide (SFLLRN, 100 μmol/L). Acetylsalicylic acid (ASA) and also the reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (100 mg over 3 days) attenuated S1P release from platelets in healthy volunteers which was paralleled by inhibition of TX formation. S1P release was increased by the thromboxane receptor (TXR) agonist U46619, and inhibited by the TXR antagonist ramatroban. After incubation of human peripheral blood monocytes with supernatants from stimulated platelets for 24 hours, chemotaxis towards serum (10%) or monocyte chemotactic protein-1 (MCP-1, 50 ng/mL) was increased about 3-fold compared to supernatants from resting platelets. This effect was inhibited when platelets were previously treated with ASA. Gene-specific knockdown suggest that the S1P receptor-1 (S1P1) and S1P3 mediate this modulation of monocyte chemotactic responses. These data suggest that S1P release from platelets after thrombin receptor activation requires TX synthesis. Inhibition of S1P release may be involved in the anti-inflammatory actions of ASA, for example by affecting recruitment of monocytes to sites of vascular injury.
- © 2010 by American Heart Association, Inc.