Background: Sphingosine 1-phosphate (S1P) is a key bioactive lipid affecting vascular tone, permeability, and hemostasis. Although multiple links between S1P and blood coagulation have been suggested, the specific mechanisms remain unclear. Thrombomodulin (TM), a crucial endothelial-bound glycoprotein, acts as an endogenous anticoagulant by converting thrombin from a procoagulant to an anticoagulant enzyme. We investigated whether S1P regulates TM expression in endothelial cells and how this interaction affects platelet adhesion and arterial thrombus formation.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in growth medium and treated with Sphingosine-1-phosphate (S1P; 1 µM) for 24 hours, with or without S1PR1 antagonist (W146) or PI3K inhibitor (LY294002) added 15 minutes before S1P incubation. TM expression was measured by ELISA. For platelet adhesion assays, HUVECs were seeded onto fibronectin-coated flow chambers, exposed to S1P, W146, or TM-antibody, and perfused with calcein-stained whole blood from healthy human volunteers. Platelet-covered areas were quantified via microscopy and image analysis. Platelet activation was analyzed in human platelet-rich plasma after S1P stimulation by measuring P-selectin expression (flow cytometry) and aggregation (aggregometry).
Wildtype (C57BL/6J) and Sphingosine-1-kinase 1-deficient (SphK1−/−) male mice (12–15 weeks) were randomly assigned to control or treatment groups, receiving S1P injections as appropriate. Aortic TM expression was determined by ELISA. Arterial thrombus formation was induced via ferric chloride injury of the carotid artery, and bleeding was assessed by tail bleeding assay. All procedures followed ethical guidelines.
Results: S1P significantly increased endothelial TM expression in HUVECs, which was abolished by S1PR1 or PI3K inhibition. S1P reduced platelet adhesion to endothelial monolayers, an effect reversed by S1PR1 blockade or TM antibody. S1P did not directly affect platelet activation; P-selectin expression and platelet aggregation remained unchanged in the absence of endothelial cells. In patients with cardiovascular disease, higher plasma S1P levels were associated with lower circulating thrombin.
In mice, S1P administration elevated TM expression in aortic endothelium and reduced arterial thrombus formation, without increased bleeding risk. SphK1-deficient mice, which have approximately 70% lower S1P, exhibited lower aortic TM expression and increased thrombus formation; TM treatment reversed this phenotype.
Conclusion: Our findings identify a novel mechanistic link between S1P signaling and endothelial thromboresistance. S1P upregulates TM expression through S1PR1 activation, leading to reduced platelet adhesion and inhibition of thrombus formation. These results highlight S1P–TM signaling as an important antithrombotic pathway in vascular homeostasis and suggest that modulation of S1P levels or TM expression could represent a potential therapeutic strategy to counteract pathological thrombosis.
