Novel actions of acetylsalicylic acid for the releases of sphingosine-1-phosphate from human platelets

Böhm, Andreas, Ulrych, Thomas , Dr. Rosenkranz, Anke C., Prof. Dr. Hohlfeld, Thomas , Dr. Rauch, Bernhard H., Prof. Dr. Schrör, Karsten. Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie, Universitätsstr. 1, 40225 Düsseldorf, Germany.

The sphingosine-derived lipid signaling molecule sphingosine-1-phosphate (S1P) is released from platelets by thrombin or selective activating-peptides for the protease-activated receptor-1 (PAR1-AP). The present study determines the effect of acetylsalicylic acid (ASA, aspirin) and the role of thromboxane formation for the release of S1P from human platelets and the functional consequences.

S1P synthesis and release were analyzed in platelets from healthy donors spiked with [3H]sphingosine. Lipids were separated by thin-layer chromatography and by mass spectrometry. Migration of human umbilical vein endothelial cells (HUVEC) and monocytic U937 cells was determined in Boyden chamber assays.

After oral application of ASA (500 mg single dose) a reduced release of S1P (by about 80%) from platelets of healthy volunteers was observed ex vivo. This effect occurred within 15 minutes after oral ASA application and persisted for 3 days until platelet TX formation started to recover. In vitro, the release of S1P from platelets which were activated by PAR1-AP (100 µmol/L) was suppressed by 80-90% by acetylsalicylic acid (ASA) (30 – 300 µmol/L) and by reversible COX inhibitors such as ibuprofen and diclofenac. The thromboxane receptor (TXR) agonist U46619 (10 µmol/L) increased S1P release 5-fold compared to untreated controls while the TXR antagonist ramatroban (2 µmol/L) inhibited it. Supernatants from PAR1-AP-activated platelets after pretreatment with ASA elicited 30% lower HUVEC and 60% lower monocyte chemotaxis as compared to supernatants from untreated positive controls. The use of specific S1P receptor inhibitors suggested this effect was S1P-dependent.

In conclusion, ASA inhibits the release of S1P from human platelets and this is dependent on TX formation and TXR activation after stimulation of PAR-1. This novel pathway may contribute to anti-angiogenic and anti-inflammatory actions of ASA by affecting recruitment of endothelial cells and monocytes to sites of injury.