Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

Figures

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  Figure 1. Structure of the compounds used in the study. The marked site shows the modification of the parent compound uridine triphosphate (UTP). Red circle mark the modified position in the original compound 1.

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  Figure 2. UTP thio-analogues antagonise adenosine diphosphate (ADP)-induced platelet aggregation. Representative tracing of platelet aggregation induced by ADP (10 µM) in the absence or presence of: 2S-UTP (A); 4S-UTP (B); 4S-isobutyl UTP (C); and UTPγS (D). Representative tracings from four experiments using independent PRP preparations. (E) Representative tracing of platelet aggregation induced by collagen (2 µg/mL) in the absence or presence of 4S-UTP (25 µM).

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  Figure 3. (A) Competitive binding of [3H]-ADP to platelet P2Y12 receptor. Platelets were incubated with 10 nM of [3H]-ADP in the absence (C; control) or presence of saturated concentrations (5x the respective IC50 values) of UTP, 2S-UTP, 4S-UTP, UTPγS and binding of [3H]-ADP to P2Y12 receptor was analysed as described in Section 4.4. AR-C66096 (AR-C; 10 µM; a potent P2Y12 receptor antagonist) was used as positive control. The data are means ± S.E.M of three experiments using independent platelet preparations. * p < 0.05 vs. control. n.s.: not significantly different from control; (B) Effect of UTP thio-analogues on cAMP level in PGE1- and ADP-stimulated human platelets. Different preparations of washed platelets were pre-incubated with PGE1 (1 µM) and UTP analogues (UTP 250 µM; 2S-UTP 100 µM; 4S-UTP 25 µM; and UTPγS 100 µM) for 10 min as indicated followed by stimulation with ADP (10 µM). * p < 0.05 vs. control, # p < 0.05 vs. PGE1, § p < 0.05 vs. ADP; (C) Effect of 4S-UTP on PGE1-mediated VASP phosphorylation (pVASP-Ser157) in ADP-stimulated human platelets. PRP was pre-incubated with vehicle (resting) or PGE1 (1 µM) followed by treatment with ADP (10 µM) or ADP plus 4S-UTP (25 µM) and…

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  Figure 4. Effect of UTP thio-analogues on ADP-induced platelet shape change. Quantification of the platelet shape change data from three independent experiments. Platelet were pre-incubated with vehicle or MRS2500 (P2Y1 receptor antagonist; 1 µM), UTP (250 µM), 2S-UTP (100 µM), 4S-UTP (25 µM), or UTPγS (100 µM) and then treated with ADP (1 µM). * p < 0.05 vs. ADP alone.