S1PR₁-signaling reduces arterial thrombosis via enhanced endothelial thrombomodulin expression

Marcel Benkhoff (Düsseldorf)1, P. Mourikis (Düsseldorf)1, M. Barcik (Düsseldorf)1, J. Dahlmanns (Düsseldorf)1, B. Knoop (Düsseldorf)1, K. Trojovsky (Düsseldorf)1, S. Ahlbrecht (Düsseldorf)1, S. Metry (Düsseldorf)1, C. Helten (Düsseldorf)1, L. K. Dannenberg (Düsseldorf)1, T. Hohlfeld (Düsseldorf)2, T. Zeus (Düsseldorf)1, M. Kelm (Düsseldorf)1, B. Levkau (Düsseldorf)3, A. Polzin (Düsseldorf)1

1Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; 2Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie Düsseldorf, Deutschland; 3Universitätsklinikum Düsseldorf Institut für Molekulare Medizin III Düsseldorf, Deutschland


Background: Sphingosine 1-phosphate (S1P) is an important lipid mediator in the cardiovascular system. Numerous links between S1P and blood coagulation have been described. However, results regarding the role of S1P in coagulation are inconclusive. A physiological antithrombotic regulator expressed by endothelial cells (EC) is thrombomodulin (TM). To date, the impact of S1P on TM-expression is unknown.

Methods: We evaluated S1P effects on TM-expression and subsequent platelet adhesion and thrombus formation. For this, we conducted cell culture with human umbilical cord venous endothelial cells (HUVECs), flow cytometry and flow chamber experiments. Additionally, in vivo arterial thrombus formation in mice was analyzed.

Results: Sphingosine kinase-1 deficient mice (SphK1-/-) have about 70% less circulating plasma S1P and show a significant reduction in TM expression on the aorta as compared to their littermates (WT: 207.2 ± 45.8 pg/g Aorta vs. SphK1-/- 124.5 ± 40.2 pg/g Aorta, p<0.0001). In contrast, S1P incubation of 24 hours significantly increased TM-expression on HUVECs by about 30 percent. This effect was abolished by S1PR1-inhibition with W146. Treatment with S1P reduced platelet adhesion from whole blood to endothelial cells in the flow chamber experiments (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). This effect could be reversed both by S1PR1 inhibition and with a TM-neutralizing antibody. P-selectin-expression on platelets was decreased after incubation with TM suggesting (Ctrl: 62.79 ± 13.30% vs. TM: 43.47 ± 11.36%, p=0.0222). Finally, to assess the in vivo effects of our findings, arterial thrombus formation in mice was performed. TM inhibited in vivo thrombus formation. Occlusion of the carotid artery could not be detected during the 30 minutes recording. In contrast, SphK1-/- mice with low S1P levels showed decreased times to first occlusion. Treatment with TM in these animals was able to reverse this effect

Conclusion: We can conclude that the main findings of our study were (i) S1P increased endothelial TM-expression, (ii) which subsequently leads to reduced platelet adhesion and (iii) inhibition of arterial thrombus formation in vivo.


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