Sonedenoson

To address the problem of resistance to standard antiplatelet therapy in some patients, our team proposed a purinoceptor-dependent dual therapy. Its efficacy is also determined by the condition of the vascular endothelium which, by secreting numerous factors, is involved in hemostasis. Among them, thrombospondin-1 is important in the context of thrombotic events. Therefore we sought to determine if the novel dual purinoceptor-dependent concept is associated with TSP-1 changes in vascular endothelial cells.

TSP-1 expression in human microvascular endothelial cells was determined at transcriptional and protein level. We performed real-time PCR, the Western blot analysis and ELISA test. We found that TSP-1 mRNA and protein expression levels significantly changed in response to P1R agonists treatment. Furthermore, we have observed that co-administration of selective A2_AR agonists (UK-432,097 or MRE0094) with P2Y₁₂R antagonists altered TSP-1 expression levels, and the direction of these changes was not synergistic. MRE0094 applied with ARC69931MX or R-138727 increased mRNA expression from 39 to 56 or 57%, respectively (*P < 0.05 vs. MRE0094; ***P < 0.001 vs. control). Also, in the case of the P2Y₁₂R antagonists used together with UK-432,097, there was an increase from 53 to 71 and 70% (*P < 0.05 vs. UK-432,097; ***P < 0.001 vs. control). The observed trends in gene expression were reflected in the protein expression and the level of its secretion from HMEC-1.

The article presents evidence which proves that the purinoceptor-dependent concept is associated with TSP-1 changes in endothelial cells (EC). Moreover, Human Microvascular Endothelial Cells treatment applied together with agonists (MRE0094 or UK-432,097) and P2Y₁₂R antagonist did not result in any synergistic effect, implicating a possible crosstalk between G proteins in GPCRs dependent signaling. Therefore, we suggest that understanding of the specific mechanism underlying TSP-1 alterations in EC in the context of the dual purinoceptor-dependent approach is essential for antiplatelet therapies and should be the subject of future research.

Adenosine analogues have high affinity and selectivity for adenosine receptors (AR), and exhibit anti-platelet activity. Plasma proteins play an important role in the regulation of platelet function and may influence the action of anti-platelet compounds. Little is known about the interactions of AR agonists with plasma proteins. This study investigates the interplay between AR agonists and plasma proteins and the consequences of those interactions. Surface plasmon resonance was employed together with molecular docking study to determine the binding kinetics of four selected ARagonists (PSB0777, Cl-Ado, MRE0094, UK432097) to several carrier proteins and to clarify the nature of these interactions. The influence of a whole plasma and of some plasma components on the effectiveness of ARagonists in the inhibition of platelet function was assessed by flow cytometry (platelet activation) and ELISA (platelet adhesion). Plasma proteins remarkably diminished the effectiveness of ARagonists in inhibiting platelet activation and adhesion in vitro. ARagonists were found to strongly bind to human serum albumin (HSA) and the protein components of lipoproteins - apolipoproteins; HSA was essential for the binding of water-soluble PSB0777, whereas apolipoproteins were needed for interactions with poorly-water soluble compounds such as UK432097 and MRE0094. In addition, HSA was shown to significantly reduce the effectiveness of PSB0777 in inhibiting ADP-induced platelet activation. In conclusion, HSA and lipoproteins are important carriers for ARagonists, which can affect pharmacodynamics of ARagonists used as platelet inhibitors.