Isolation and identification of aptamers from defibrotide that act as thrombin antagonists in vitro.

This study was designed to isolate and identify aptamer sequences, acting as thrombin antagonists, from defibrotide, a single-stranded DNA fraction. Two different aptamers were identified: 5'-GGTTGGATTGGTTGG-3' and 5'-GGTTGGATCGGTTGG-3'. A third aptamer: 5'-GGATGGATCGGTTGG-3' was found in the PCR product from the double-stranded defibrotide precursor. All aptamers were potent inhibitors of thrombin-induced platelet aggregation, thromboxane biosynthesis, increase in cytosolic Ca++ and fibrin clot formation, effective concentrations being in the nanomolar range. There was no effect on U 46,619 (thromboxane mimetic) or collagen-induced responses. Similar effects were obtained with unfractionated defibrotide while the "nonsense" oligonucleotide 5'-GGTGGTGGTTGTGGT-3' was inactive. The data suggest that antithrombin effects of defibrotide are caused by aptamers.