Aptamer inhibits degradation of platelet proteolytically activatable receptor, PAR-1, by thrombin

We investigated the in vitro effects of the site-directed thrombin inhibito r-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTT GG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and pla telet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin -dependent degradation of proteolytically activatable receptor, PAR-1, expo sed on platelet surface membrane at micromolar concentrations. The incubati on of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up t o fourfold, P < .0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46 +/- 20% inhibition at 0.15 U/ml thrombin, P < .0 01), as well as thrombin-mediated platelet aggregation in PRP (7 +/- 10% in hibition at 1 U/ml thrombin, P < .05). Furthermore, aptamer inhibited the t hrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17 %, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 n M (P < .025 by randomised block analysis; P-regression (slope) < .0001). We conclude that aptamer is able to considerably attenuate thrombin proteolyt ic activity regardless of the molecular size of thrombin substrates. Our ob servations directly proved that aptamer may be successfully used for the in hibition of thrombin activity towards various physiological targets: one re lated to fibrin generation in the final stage of coagulation cascade, and a nother concerning the interaction of thrombin with its surface membrane rec eptor, PAR-1, in blood platelets. (C) 2001 Elsevier Science Ltd. All rights reserved.