In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits.
Using a novel in vitro selection/amplification technique, we have recently identified a new class of thrombin inhibitors based on single-stranded DNA oligonucleotides. One oligonucleotide, GGTTGGTGTGGTTGG (thrombin, aptamer), showed potent anticoagulant activity in vitro. We have initiated pharmacologic studies in cynomolgus monkeys to study the thrombin aptamer's in vivo anticoagulant properties. Upon infusion of the thrombin aptamer, anticoagulation was rapidly achieved, with a plateau reached within 10 minutes. There was a linear dose-response relationship between thrombin aptamer infusion rate and prolongation of plasma prothrombin time. Ten minutes after the infusion was stopped, no prolongation of prothrombin time was observed, indicating that the thrombin aptamer has an extremely short in vivo half-life, estimated to be 108 +/- 14 seconds. In addition, inhibition of thrombin-induced platelet aggregation in platelet-rich plasma was observed ex vivo without an effect on collagen-induced aggregation, indicating that the inhibition was specific for thrombin and not due to a nonspecific inhibitory effect on platelets. To exploit the short in vivo half-life of the thrombin aptamer, its ability to achieve regional anticoagulation in an extracorporeal hemofiltration circuit in sheep was tested. Doubling of the prothrombin time in the circuit was observed, whereas the systemic prothrombin time was minimally prolonged. We conclude that the thrombin aptamer is a potent anticoagulant in vivo, and specifically inhibits thrombin-induced platelet aggregation ex vivo. The rapid onset of action and short half-life in vivo suggest that the thrombin aptamer may be useful in anticoagulation with extracorporeal circuits and may have distinct advantages in certain acute clinical settings.
Gilead Sciences Inc., Foster City, CA 94404.
L C
LC
Griffin
G F
GF
Tidmarsh
L C
LC
Bock
J J
JJ
Toole
L L
LL
Leung
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D052157:Aptamers, Nucleotide; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry; D006207:Half-Life; D006440:Hemofiltration; D007700:Kinetics; D008252:Macaca fascicularis; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010974:Platelet Aggregation / Q000187:drug effects; D011119:Polynucleotides / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D011517:Prothrombin Time; D012756:Sheep; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000494:pharmacology
D000925:Anticoagulants; D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011119:Polynucleotides; C081676:thrombin aptamer; D013917:Thrombin