Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit a critical thiol group in the active form that binds to the P2Y₁₂ receptor to inhibit platelet activation and prevent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation (SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formation of thienopyridine-SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueous solution and adding sodium nitrite, or albumin-SNO. Ozone-based chemiluminescence techniques were utilised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregation was monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations of ticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivatives directly from anionic nitrite in water under laboratory conditions without the need for prior metabolism. Thienopyridine-SNO formation was dependent on pH, duration of mixing and nitrite concentration, with prasugrel-SNO being more favourably formed. The SNO moiety readily participated in trans-nitrosation reactions with albumin and plasma. Prasugrel-SNO showed significantly better inhibition of platelet aggregation compared with clopidogrel-SNO, however when compared on the basis of SNO concentration these were equally effective (IC₅₀=7.91 ± 1.03 v/s 10.56 ± 1.43 μM, ns). Thienopyridine-derived SNO is formed directly from the respective base drug without the need for prior in vivo metabolism and therefore may be an important additional contributor to the pharmacological effectiveness of thienopyridines not previously considered.