Page 145 - 2020_11-Haematologica-web

Novel DNA aptamer targeting VWF A1 domain
Figure 1. The inhibitory effects of TAGX-0004, ARC1779, and caplacizumab on platelet aggregation under static conditions. As shown in the upper left panel, TAGX- 0004 completely inhibited ristocetin-induced platelet aggregation (RIPA) at a concentration of 50 nM. In contrast, the concentration required for ARC1779 was 500 nM, and that for caplacizumb was 50 nM. As shown in the lower left panel, TAGX-0004 inhibited botrocetin-induced platelet aggregation (BIPA) at a concentration of 50 nM. The concentration required for ARC1779 was 500 nM, and that for caplacizumab was 50 nM.
results indicate that TAGX-0004 inhibits platelet throm- bus formation at least 20 times more potently than ARC1779 under high shear stress, and with a similar potency as caplacizumab.
Biophysical interaction analysis with EMSA
Biophysical interaction analysis with EMSA was per- formed four times for each aptamer. Figure 3 shows that the affinity of TAGX-0004 to VWF A1 domain (KD=2.2± 0.9 nM [n=4]) was approximately 16-fold higher than that of ARC1779 (KD =35.5±1.5 nM [n=4]).
Alanine scanning mutagenesis with EMSA
Figure 4 shows the results of EMSA using 16 alanine- mutated VWF A1 with TAGX-0004 (A) or ARC1779 (B). First, we analyzed nine mutants as shown in the left panels. Based on the results of this initial analysis, we then analyzed additional seven mutants as shown in the right panels. Of these, 10 VWF A1 mutants (K1312A, R1334A, R1336A, K1348A, K1371A, E1376A, K1406A, K1423A, R1426A, and K1430A) formed shifted bands representing complexes between an aptamer and VWF A1, as well as between wild-type (WT) VWF A1 and each aptamer. In the remaining six mutants, the intensity of the complexed bands was decreased, and the intensity of the unbound aptamer bands was increased. Of these mutants, R1395A and R1399A showed decreased intensity in both TAGX- 0004 and ARC1779 band complexes.
While the binding affinity to ARC1779 was signifi- cantly decreased in R1287A, K1362A, and R1392A, the
binding affinity to TAGX-0004 was decreased in F1366A. These findings suggest that R1395 and R1399 are essen- tial residues for binding to both aptamers, and that R1287, K1362, and R1392 probably contribute to binding to only ARC1779, while F1366 is required for binding to TAGX-0004. Our data indicate that both aptamers bind to the VWF A1 domain, even though the residues neces- sary for this binding are partially different, and these dif- ferences might affect their VWF inhibitory activity.
Competition assay with Biacore
To compare the binding sites of the aptamers to the VWF A1 domain, a competition assay was performed by SPR using Biacore. Biotinylated TAGX-0004 was immobi- lized on the sensor chip SA. Then, VWF A1 and a com- petitor aptamer (TAGX-0004 or ARC1779) as an analyte was injected onto the chip. The results of a self-competi- tion assay using TAGX-0004 showed that the amount of VWF A1 binding to the chip decreased depending on the concentration of the competitor TAGX-0004 (Online Supplementary Figure S1). ARC1779 also dose-dependently decreased the response unit value. These results indicate that TAGX-0004 and ARC1779 bind to the overlapped region on the VWF A1 domain. Moreover, the competitive ability of ARC1779 against immobilized TAGX-0004 on the chip surface was lower than that of TAGX-0004 against TAGX-0004. These results were consistent with those of EMSA and indicated that the binding affinity of TAGX-0004 for VWF A1 was higher than that of ARC1779.
haematologica | 2020; 105(11)
2633