15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and sphingosine-1-phosphate

Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood.. The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis.. S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays.. Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 μM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets.. TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Blood Platelets; Cells, Cultured; Chromatography, Thin Layer; Humans; Lysophospholipids; Receptors, Thromboxane; Sphingosine; Thrombin; Thromboxanes

We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Calcium; Calcium Signaling; Collagen; Crotalid Venoms; Humans; Lectins, C-Type; Lysophospholipids; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet Membrane Glycoproteins; Receptors, Thrombin; Sphingosine; Tyrosine

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Calcium Signaling; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sphingosine; Thrombin