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K. Sakai et al.
thrombosis. We recently generated a novel DNA aptamer, TAGX-0004, that contains the artificial hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds); this aptamer tar- gets human VWF A1 with very high affinity (KD = 61.3 pM) and specificity.14
In this study, we compared TAGX-0004, ARC1779, and caplacizumab in terms of their inhibitory effects on VWF activity, by performing conventional platelet aggregation assays, ristocetin- and botrocetin-induced platelet aggrega- tion assays (RIPA and BIPA, respectively), and shear-stress– induced aggregation assays using the microchip flow cham- ber system (T-TAS®).15 To assess the affinity and binding sites of the two aptamers, we performed biophysical inter- action analysis and alanine scanning mutagenesis of the VWF A1 domain. In addition, we analyzed the binding sites of caplacizumab using surface plasmon resonance (SPR).
Methods
Details of the materials and methods are presented in the Online Supplementary Materials and Methods section.
Sources of nucleoside, oligonucleotides, nanobody, and protein
The artificial nucleoside phosphoramidite (dDs-CE Phosphoramidite) was synthesized as described previously.16,17 The oligonucleotides TAGX-0004 and ARC1779 (without poly- ethylene glycol [PEG]) were synthesized by and purchased from GeneDesign, Inc. (Osaka, Japan). The oligonucleotide sequence of TAGX-0004 (as Rn-DsDs-51mh2)14 and that of ARC177918 were reported previously. The anti-human VWF nanobody caplacizum- ab (as TAB-234) was purchased from Creative-Biolabs (New York, NY, USA). The recombinant human VWF A1 domain protein was purchased from U-Protein Express BV (Utrecht, the Netherlands), and was used for the measurement of the binding affinity to anti- human VWF A1 agents using SPR.
Alanine-scanning mutagenesis
Based on previous reports that described the interaction between VWF A1 and GPIb,19 botrocetin,20 or ARC1172,21 we designed 16 alanine-substituted mutants of the human VWF A1 domain (R1287, K1312, R1334, R1336, K1348, K1362, F1366, K1371, E1376, R1392, R1395, R1399, K1406, K1423, R1426, and K1430). These mutant proteins were generated using a cell-free expression system (Taiyo Nippon Sanso Corporation, Tokyo, Japan).
Platelet aggregation test
A platelet aggregation test (PAT) was performed with PRP313M aggregometer (TAIYO Instruments INC, Osaka, Japan). Aggregation inducing substances and their final concentration were as follows; ristocetin (1.5 mg/mL), botrocetin (1.0 mg/mL), collagen (4 mg/mL), epinephrine (1.0×10-4 M) and adenosine diphosphate (ADP) (1.0×10-5 M).
Total thrombus formation analysis system
The total thrombus formation analysis system (T-TAS®) (Zacros, Fujimori Kogyo Co. Ltd., Tokyo, Japan) is a micro-chip flow-chamber device used to visually and quantitatively analyze thrombus formation in whole blood samples under various blood flow conditions.15 Whole blood samples with each anti-VWF A1 agent were applied onto a collagen I coated micro-chip (PL chip). Subsequently, the thrombus formation in the capillaries and an increase in flow pressure were observed.
Electrophoresis mobility shift assay
The binding abilities of the two aptamers to the human VWF A1 domain and its alanine-substituted mutants were analyzed by electrophoresis mobility shift assay (EMSA). Each aptamer (final concentration of 100 nM) was mixed with VWF A1 (final concen- tration of 0-800 nM) in binding buffer and incubated at 37 °C for 30 minutes (min), then subjected to 8% native PAGE in 0.5× TBE buffer for 50 min at room temperature (200 V/cm). The aptamer- VWF A1 complexes were detected as a shift band, and the band patterns were detected by SYBR Gold. The dissociation rate (KD) was calculated by Scatchard plot analysis.
Surface plasmon resonance
Competition assays with the two aptamers were performed by surface plasmon resonance (SPR) analysis using Biacore T200 (GE Healthcare UK Ltd., Little Chalfont, UK), as described previously.14
We also performed SPR analysis to investigate the binding site of the VWF A1 domain to caplacizumab.
Structure models of the VWF A1 domain
Three-dimensional (3D) structure models of the VWF A1 domain were visualized with the PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, USA).
Ethical statement
This study was approved by the ethics committee of Nara Medical University. Written informed consent was obtained from the individual who donated plasma for the use in this study.
Results
Inhibitory effects on platelet aggregation
The inhibitory effects of TAGX-0004, ARC1779, and caplacizumab under static conditions were analyzed by PAT. Figure 1 shows representative results of the analyses of the three types of plasma. All three agents showed inhi- bition activities in both RIPA and BIPA. However, the inhibitory effects on platelet aggregation differed between the three agents. In RIPA, the 80% maximal inhibitory concentration (IC80) of TAGX-0004 was 50 nM, whereas that of ARC1779 was 500 nM and that of caplacizumab was 50 nM; thus TAGX-0004 blocked VWF function via the A1 domain approximately 10 times more potently than ARC1779, and exhibited a similar potency as capla- cizumab. In BIPA, the IC80 of TAGX-0004 was 50 nM, whereas that of ARC1779 was 500 nM and that of capla- cizumab was 50 nM, respectively; thus TAGX-0004 blocked VWF function approximately 10 times more potently than ARC1779 showing a similar potency as caplacizumab. None of the three agents inhibited platelet aggregation induced by collagen, epinephrine, or ADP (data not shown).
Inhibitory effects on thrombus formation
To assess the inhibitory effect of the three agents on platelet thrombus formation, we performed T-TAS. In this study, complete inhibition was defined as an increase in flow pressure from baseline of no more than 10 kPa. TAGX-0004, ARC1779, and caplacizumab were analyzed three times each, and Figure 2 shows representative flow pressure curves. Both TAGX-0004 and caplacizumab pre- vented thrombus occlusion under the flow condition at 50 nM. In contrast, ARC1779 did not demonstrate complete inhibition, even at a concentration of 1,000 nM. These
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haematologica | 2020; 105(11)