Inhibitory Effect of a Novel DNA Aptamer with Artificial Nucleic Bases for Von Willebrand Factor A1 Domain on Platelet Thrombosis Formation

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction with its A1 domain and platelet glycoprotein 1b. Thus, VWF A1 domain was thought to be a good therapeutic target candidate for VWF mediated thrombosis. In this study, we analyzed the inhibitory effects of a novel DNA aptamer (TAGX-0004) on platelet aggregation compared with another VWF A1 domain binding aptamer (ARC1779) which had entered to Phase II clinical trial for acquired thrombotic thrombocytopenic purpura (TTP) in 2011. TAGX-0004 was generated by SELEX (systematic evolution of ligands by exponential enrichment) and consisted of artificial nucleic acid base, 7-(2-thienyl) imidazo [4,5-b] pyridine (Ds) as well as natural bases (Adenine, Thymine, Cytosine and Guanine). The dissociation constant (Kd) of the aptamers was analyzed by electrophoretic mobility shift assay (EMSA). 100 nM of each DNA aptamer was mixed with A1 domain of VWF protein (final concentration was 0 to 800 nM) in binding buffer (1x PBS, 0.005% NP-40) and incubated for 30 min at 37 °C. The samples were subjected to EMSA with 8% native PAGE, then stained by SYBR Gold. Kd value was determined with scatchard plot analysis. To characterize the binding sites of VWF A1 to these DNA aptamers, various mutants of VWF A1 domain were generated with an alanine substitution technique. Platelet aggregation was induced with ristocetin (RIPA), botrocetin (BIPA), collagen, epinephrine and adenosine diphosphate (ADP). The change of light transmission rate of platelet rich plasma (PRP) compared to platelet poor plasma (PPP) at 37 °C was recorded for 6 min, then the inhibition ratio of platelet aggregation was determined. Total thrombus-formation analysis system (T-TAS) (Fujimori Kogyo Co. Ltd., Tokyo, Japan), which is a novel micro-chip flow-chamber system, was employed to analyze thrombus formation visually and quantitatively in whole blood samples. The micro-chip coated with type 1 collagen was used for this analysis. Anti-VWF A1 inhibitory effects of these aptamers at high shear stress (initial rate of 2000 s-1) were calculated by continuous pressure levels within its narrow capillary. The course of thrombus formation was also optically recorded with a video-microscope located under the microchip. Biophysical interaction analysis using EMSA showed that TAGX-0004 had approximately 15-times higher binding activity to VWF A1 domain than ARC1779. Based on an alanine scan ana