Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Impact of sphingosine-1-phosphate receptor 1 (S1PR1) activation on platelet reactivity
C. Tolksdorf1, R. Moj2, A. Šimunović2, R. Wolf2, E. Moritz2, G. Jedlitschky2, B. H. Rauch1
1Abteilung Pharmakologie und Toxikologie, Carl von Ossietzky Universität Oldenburg, Oldenburg; 2Institut für Pharmakologie, Universitätsmedizin Greifswald, Greifswald;

Background:

Sphingosine-1-phosphate (S1P) is a bioactive phospholipid which is known to affect not only cell migration, adhesion and survival but also key processes of hemostasis. In platelets, S1P is formed from sphingosine by sphingosine kinase 2 (SphK2). Platelets store and upon activation release large amounts of S1P and are considered as a main source of circulating S1P. After its release, S1P exerts its effects through binding in an autocrine or paracrine manner to five G-coupled receptors: the S1PRs 1-5. However, the precise role of each S1PR is to date unclear. In this study, we focused on the function of the S1PR1 for platelet aggregation. This may be particularly relevant for diseases with elevated platelet reactivity, such as the coronary heart disease.

Purpose: The effects of S1P on the primary hemostasis are poorly understood. Therefore, we investigated the impact of S1PR1 activation on platelet aggregation with specific S1PR1-agonists and -antagonists.

Methods:

Peripheral venous blood was collected from healthy volunteers after written informed consent. Expression and potential co-localization of S1PR1 and SphK2 in platelets was determined by immunofluorescence microscopy. Protein expression was also determined by western blotting. For platelet aggregation studies using light transmission aggregometry (LTA) platelet rich-plasma (PRP) was generated by low-speed-centrifugation. PRP was incubated with the respective S1PR1-agonist (CYM5442) or -antagonist (EX26) concentrations. Subsequently, platelet aggregation was induced by addition of adenosine 5’-diphosphate (ADP), epinephrine or collagen in various concentrations to evaluate potential synergistic or antagonistic effects.

Results:

Expression of both S1PR1 and SphK2 in platelets was verified by immunofluorescence microscopy and western blotting. No effect on platelet aggregation was detected when PRP was stimulated with S1PR1-agonist CYM5442 or -antagonist EX26 (10 µM) alone. However, pre-incubation with the S1PR1-agonist maintains and even elevates ADP-induced platelet aggregation for up to 1.5 hours. This effect was reversed by co-incubation with the S1PR1-antagonist Ex26 and only observed for CYM5442 with an ADP-concentration of 10 µM. Platelet aggregation induced by 10 µM (but not 5 µM) epinephrine did slightly but not significantly increase maximum platelet aggregation. No additional effect on platelet aggregation was seen after stimulation of S1PR1 and subsequent platelet activation with 1,5 µg/mL collagen. Furthermore, regarding slope or lag-time of platelet-aggregation, no effect of the S1PR1 on platelet aggregation was detected.

Conclusion:

Synergistic activation of the S1PR1 and ADP 10 µM maintains platelet reactivity over time. This effect was only detected with ADP in a concentration dependent manner. Therefore, modulating the S1P-S1PR1 pathway in platelets may provide a novel therapeutic strategy for cardiovascular patients with hyperactive platelets and eventually pathologically elevated S1P levels.


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