Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Objective: To compare the prophylactic and thrombolytic effects of RNA aptamer (9.14T79VRT7) on ex vivo canine platelet function. Approach and Results: We previously demonstrated that inhibition of von Willebrand Factor (VWF) by a targeted RNA aptamer prevents thrombosis and thrombolyses stabilized clots in a FeCl 3 -induced murine vascular injury model suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesize that 9.14T79VRT7, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA in murine and canine thrombotic models, may affect additional agonist pathways to mitigate platelet activation, aggregation, and adhesion. Platelet aggregation before and after 9.14T79VRT7 addition was analyzed utilizing WB aggregometry based on impedance in five adult beagles. Agonist concentrations were selected from previous publications which had optimized platelet reactivity in canine WB and included: collagen (3.2 ug/ul), ADP (20 uM), arachidonic acid (0.5 mM), and botrocetin (1 ug/ul). The 9.14T79VRT7 concentration used (25 nM) was selected as the most effective as assessed by Total Thrombus Analysis System (TTAS). Botrocetin elicited the greatest response before 9.14T79VRT7 addition, followed by Collagen, ADP, and Arachidonic acid. Impedance resulting from prophylactic addition of 9.14T79VRT7 before Collagen, ADP, and Botrocetin resulted in a 50.00%, 34.83%, and 38.26% change in amplitude, respectively and a 43.70%, 14.28%, and 35.14% change in slope, respectively (p<.0001). There was no significant difference in response after Arachidonic acid. Impedance resulting from thrombolytic addition of 9.14T79VRT7 after agonist response to Collagen, ADP, and Botrocetin resulted in a 78.66%, 94.08%, and 84.58% change in amplitude, respectively and a 67.28%, 61.28%, and 63.73% change in slope, respectively (p<.0001). Although amplitude and slope decreased with 9.14T79VRT7 addition after Arachidonic acid, the change was not significant. Both control groups (9.14T79VRT7 and platelet buffer diluent) resulted in no significant changes. Conclusion: 9.14T79VRT7 markedly changes platelet response both before and after agonist treatment in canine ex vivo impedance analysis.