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

Abstract <Introduction> 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. <Methods> 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. <Results> 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 analysis, it was revealed that a couple of residues we tested were critical for binding of ARC1779 but not for TAGX-0004, which indicating that TAGX-0004 interacts with VWF A1 domain via different amino acid residue(s). Both aptamers did not inhibit the platelet aggregation induced by collagen, epinephrine or ADP. In RIPA, the 80% inhibition was observed by TAGX-0004 and ARC1779 at a final concentration of 10 nM and 750 nM, respectively. In BIPA, those were seen at those of 50 nM and 1000 nM, respectively. In T-TAS analysis, TAGX-0004 inhibited the thrombus formation completely at a final concentration of 100 nM, whereas ARC1779 exhibited partial inhibition of thrombosis formation even at 1000 nM. These results indicated that TAGX-0004 had stronger inhibitory effect on the platelet aggregation compared with ARC1779 under both static and high-shear conditions. <Conclusions> In the present study, we showed the affinity to VWF-A1 domain of TAGX-0004 was stronger than that of ARC1779. In addition, TAGX-0004 was more effective in suppressing platelet aggregation under both static and high-shear stress condition than ARC1779. In the published results of clinical study of ARC1779, there were no hemorrhagic complications in the small clinical trial of patients (7 patients of ARC1779 group) despite the almost complete suppression of VWF function in severely thrombocytopenic patients. Therefore, TAGX-0004 could be developed as a promising therapeutic agent for VWF mediated thrombotic disorders, such as acute coronary syndrome and TTP. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

Blood ◽

10.1182/blood.v95.12.3796 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3796-3803 ◽

Cited By ~ 16

Author(s):

Nadine Ajzenberg ◽

Anne-Sophie Ribba ◽

Ghassem Rastegar-Lari ◽

Dominique Meyer ◽

Dominique Baruch

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Von Willebrand Disease ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Consistent Increase ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Download Full-text

Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of αIIbβ3 engagement

Blood ◽

10.1182/blood-2002-06-1879 ◽

2003 ◽

Vol 101 (11) ◽

pp. 4363-4371 ◽

Cited By ~ 6

Author(s):

Médina Mekrache ◽

Christilla Bachelot-Loza ◽

Nadine Ajzenberg ◽

Abdelhafid Saci ◽

Paulette Legendre ◽

...

Keyword(s):

Shear Rate ◽

Von Willebrand Factor ◽

Thrombus Formation ◽

High Shear Rate ◽

Von Willebrand Disease ◽

High Shear ◽

High Level ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Abstract Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.

Download Full-text

Identification of the Binding Site for an Alloantibody to von Willebrand Factor which Inhibits Binding to Glycoprotein Ib within the Amino-terminal Region Flanking the A1 Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614572 ◽

1999 ◽

Vol 81 (05) ◽

pp. 793-798 ◽

Cited By ~ 6

Author(s):

Masaru Shibata ◽

Yoshihiro Fujimura ◽

Yukihiro Takahashi ◽

Hiroaki Nakai ◽

Yoshihiko Sakurai ◽

...

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Von Willebrand Disease ◽

Tryptic Fragment ◽

Amino Terminal ◽

A1 Domain ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

SummaryAn alloantibody to von Willebrand factor (vWF) which developed in a Japanese boy with type 3 von Willebrand disease has been characterized. The antibody was non-precipitating IgG and the main subclasses were IgG2 and IgG4. The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its predominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryptic fragment of vWF (residues 449-728). No reaction was seen, however, with either a 39/34 kDa dispase fragment (480-718) or a recombinant vWF fragment (residues 465-728). These findings suggested that the essential epitope resided in the amino-terminal flanking region of the A1 domain. We synthesized overlapping peptides corresponding to the region containing D3/A1 boundary. A peptide, residues 458-472, bound to the antibody and dose-dependently blocked the antibody binding to the 52/48 kDa fragment. The same peptide neutralized the inhibitory effect of the alloanti-body on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which reacted with the 52/48 kDa fragment.Furthermore, the specific component of the antibody, directed against residues 458-472, blocked vWF binding to GPIb in absence of exogenous agonist. Our results suggest that the region flanking the A1 domain plays an important role in regulating vWF binding to GPIb.

Download Full-text

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions

Thrombosis and Haemostasis ◽

10.1160/th09-01-0052 ◽

2009 ◽

Vol 102 (09) ◽

pp. 529-537 ◽

Cited By ~ 1

Author(s):

Zhou Zhou ◽

Aubrey Bernardo ◽

Qiqing Zhu ◽

Yongli Guan ◽

Wensheng Sun ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Flow Conditions ◽

Β Sheets ◽

A1 Domain ◽

Von Willebrand ◽

Potential Interactions ◽

Willebrand Factor ◽

Binding Subunit

SummaryPlatelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury.This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets.However,the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibα, theVWF-binding subunit of the GP Ib-IX-V complex.We evaluated the potential interactions of T30923 with GP Ibα and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the β-sheets of the N-terminal region of GP Ibα (E14-D18, S39, D63-S64, and D83-S85). Experimentally,T30923 bound GP Ibα and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilized VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP IbαVWF interaction.

Download Full-text

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

Blood ◽

10.1182/blood.v95.12.3796.012k05_3796_3803 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3796-3803 ◽

Cited By ~ 1

Author(s):

Nadine Ajzenberg ◽

Anne-Sophie Ribba ◽

Ghassem Rastegar-Lari ◽

Dominique Meyer ◽

Dominique Baruch

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Von Willebrand Disease ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Consistent Increase ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Download Full-text

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Download Full-text

Signaling through GP Ib-IX-V activates αIIbβ3 independently of other receptors

Blood ◽

10.1182/blood-2003-10-3664 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3403-3411 ◽

Cited By ~ 120

Author(s):

Ana Kasirer-Friede ◽

Maria Rita Cozzi ◽

Mario Mazzucato ◽

Luigi De Marco ◽

Zaverio M. Ruggeri ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Signaling Proteins ◽

Phosphatidylinositol 3 Kinase ◽

Kinase C ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor ◽

Pharmacologic Inhibition

Abstract Platelet adhesion to von Willebrand factor (VWF) activates αIIbβ3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates αIIbβ3 activation directly or through other signaling proteins physically associated with it (eg, FcR γ-chain), possibly with the contribution of other agonist receptors and of VWF signaling through αIIbβ3. To resolve this question, human and GP Ibα transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain and the adapter molecule, ADAP, and triggered intracellular Ca2+ oscillations and αIIbβ3 activation. Inhibition of Ca2+ oscillations with BAPTA-AM prevented αIIbβ3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented αIIbβ3 activation but not Ca2+ oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibα transgenic platelets lacking FcR γ-chain. These data establish that GP Ib-IX-V itself can signal to activate αIIbβ3, through sequential actions of Src kinases, Ca2+ oscillations, and PI 3-kinase/PKC. (Blood. 2004;103:3403-3411)

Download Full-text

Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor

Blood ◽

10.1182/blood.v91.10.3792.3792_3792_3799 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3792-3799 ◽

Cited By ~ 3

Author(s):

Hilde Depraetere ◽

Nadine Ajzenberg ◽

Jean-Pierre Girma ◽

Catherine Lacombe ◽

Dominique Meyer ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Platelet Glycoprotein ◽

Rotational Viscometer ◽

Induce Platelet Aggregation ◽

Static Conditions ◽

Von Willebrand ◽

Willebrand Factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.

Download Full-text

Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab

Haematologica ◽

10.3324/haematol.2019.235549 ◽

2019 ◽

Vol 105 (11) ◽

pp. 2631-2638 ◽

Cited By ~ 4

Author(s):

Kazuya Sakai ◽

Tatsuhiko Someya ◽

Kaori Harada ◽

Hideo Yagi ◽

Taei Matsui ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Von Willebrand Factor ◽

Plasmon Resonance ◽

Binding Sites ◽

Thrombus Formation ◽

Static Conditions ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

Download Full-text