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.
Bracht
F
F
Institut für Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Schrör
K
K
D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000097:blood; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D011089:Polydeoxyribonucleotides / Q000235:genetics* / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors*
D004277:DNA, Single-Stranded; D010975:Platelet Aggregation Inhibitors; D011089:Polydeoxyribonucleotides; C036901:defibrotide; D013917:Thrombin; D002118:Calcium