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K. Sakai et al.
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Figure 4. Electrophoresis mobility shift assay (EMSA) using alanine-scanning mutants for analyzing von Willebrand factor A1 binding to TAGX-0004 and ARC1779.
The white arrowhead indicates the complex of von Willebrand factor (VWF) A1 with an aptamer (TAGX-0004 or ARC1779). The black arrowhead indicates unbound aptamer. The leftmost lane in each gel (-) represents aptamer only, without a VWF A1 mutant. Using both aptamers, 10 VWF A1 mutants (K1312A, R1334A, R1336A, K1348A, K1371A, E1376A, K1406A, K1423A, R1426, and K1430A) formed complexes with aptamers, as did wild-type (WT) VWF A1. With TAGX-0004, as shown in the top panels, three mutants (F1366A, R1395A, and R1399A, indicated in red letters) demonstrated decreased densities of complex bands and increased densities of unbound bands, indicating that these amino acids were important for the binding of VWF A1 to TAGX-0004. With ARC1779, as shown in the lower panels, five mutants (R1287A, K1362A, R1392A, R1395A, and R1399A indicated in red letters) demonstrated decreased densities of complex bands and increased densities of unbound bands.
and botrocetin, the minimum concentration of TAGX- 0004 necessary for inhibition is significantly lower than that of ARC1779 in both RIPA and BIPA (Figure 1). PAT analysis revealed that TAGX-0004 can block VWF func- tion via the A1 domain at least 10 times more strongly than ARC1779. T-TAS also revealed that TAGX-0004 is superior to ARC1779 at inhibiting VWF function (Figure 2). In addition, compared with caplacizumab, TAGX-0004 showed equally effective inhibition against thrombosis formation under various blood flow conditions. Moreover, TAGX-0004 has a unique mini-hairpin DNA structure that offers benefits in pharmaceutical applica- tions,14 specifically by conferring resistance to degradation by nucleases. This structure should extend the half-life of this molecule in vivo.
As described above regarding the adverse effects of caplacizumab treatment, there are still concerns regarding bleeding caused by anti-VWF antagonists. Although anti- VWF agents have demonstrated superior safety profiles in this regard compared to anti-platelet agents, measures to quickly treat bleeding that occurs during aptamer treat- ment should be prepared. We speculate that if a neutralizing agent comprising a DNA sequence that is par- tially complementary to that of a specific aptamer is pre- pared, it may serve as an effective antidote as introduced in the literature.29 In the case of TAGX-0004, such a partial DNA sequence may contain pyrrole-2-carbaldehyde (Pa), the complementary base pair to Ds.16 In the past develop- ment of antithrombotic aptamer therapeutics, similar approaches of antidotes were taken to neutralize the
unwanted effect of an aptamer, even though such aptamers are still to be approved.26 The specific antidote could contribute to control severe bleeding compared to caplacizumab.
Alanine mutagenesis analysis of the VWF A1 domain was performed in this study to determine the binding sites of TAGX-0004, ARC1779, and caplacizumab. The binding sites of ARC1779, but not TAGX-0004 or caplacizumab, were reported previously.21 Our results show that the bind- ing sites of these three agents only partially correspond to each other (Figure 6). Huang et al.21 reported that 18 amino acid residues in the VWF A1 domain were binding sites for ARC1172, which has the same fundamental structure as ARC1779. That study did not identify R1399 as a binding site, even though in the present study it is a common bind- ing site for all three agents. Chen et al. identified R1399 as one of the key amino acids for the effect of VWF to hemo- stasis and thrombosis.30 Interestingly, a hydrophobic amino acid residue (F1366) is required to bind to TAGX-0004, which has two hydrophobic Ds bases. Matsunaga et al. pre- viously reported that the number of Ds bases (0-2) strongly correlated with binding affinity to VWF A114 and we con- firmed that Ds-free TAGX-0004 failed to inhibit thrombus formation on T-TAS (data not shown). These results indicate that the base of Ds interacts directly with the VWF A1 domain and therefore F1366 appears to be an essential and unique binding site of TAGX-0004 to the VWF-A1 domain with hydrophobic interaction (e.g., pi-stacking). Of note, alanine scanning of caplacizumab showed that like ARC1779 and TAGX-0004, both R1395 and R1399 are nec-
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haematologica | 2020; 105(11)