Platelet P2Y₁₂ is an important ADP receptor that is involved in agonists-induced platelet aggregation and is an important target for the development of anti-platelet aggregation drugs. Here the effects of thio-analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation are characterised. Using human platelet rich plasma we demonstrate that UTP inhibits P2Y₁₂ but not P2Y₁ receptors and antagonises ADP-induced platelet aggregation in a conc.-dependent manner with an IC₅₀ value of ~250 mM against ADP (10 mM). An 8-fold increase in the platelet inhibitory activity was observed with 2-thio analogue of UTP (2S-UTP) with an IC₅₀ value of 30 mM. A 33-fold increase in anti-platelet aggregation activity was observed with 4-thio analogue (4S-UTP) with an IC₅₀ value of 7.5 mM. However, a 3-fold decrease in activity was observed by introducing an isobutyl group at the 4S- position. A complete loss in anti-platelet aggregation activity was observed with thio-modification of gamma phosphate of the sugar moiety which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y₁₂ receptors was further verified by P2Y₁₂ receptor binding assay and cAMP assay. The novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y₁₂ receptor antagonists that can be useful candidates for therapeutic intervention.