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

Haematologica ◽  
2019 ◽  
Vol 105 (11) ◽  
pp. 2631-2638 ◽  
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.

Development and Applications of Surface Plasmon Resonance-Based von Willebrand Factor– Collagen Binding Assay

2002 ◽  
Vol 302 (2) ◽  
pp. 252-262 ◽  
Author(s):  
Evgueni Saenko ◽  
Christoph Kannicht ◽  
Klemens Loster ◽  
Andrey Sarafanov ◽  
Alexey Khrenov ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Von Willebrand Factor ◽  
Plasmon Resonance ◽  
Binding Assay ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

The A/T1381 polymorphism in the A1-domain of von Willebrand factor influences the affinity of von Willebrand factor for platelet glycoprotein Ibα

2007 ◽  
Vol 98 (07) ◽  
pp. 178-185 ◽  
Author(s):  
Tímea Szántó ◽  
Ágota Schlammadinger ◽  
Stephanie Staelens ◽  
Simon De Meyer ◽  
Kathleen Freson ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Association Studies ◽  
Platelet Glycoprotein ◽  
Platelet Receptor ◽  
Increased Risk ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMany polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphis mA/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbα. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbα under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbα than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbα, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events.

Regulation of von Willebrand factor-platelet interactions

2010 ◽  
Vol 104 (09) ◽  
pp. 449-455 ◽  
Author(s):  
Julie Pegon ◽  
Evelyn Groot ◽  
Philip de Groot ◽  
Peter Lenting
Keyword(s):  
Von Willebrand Factor ◽  
Thrombus Formation ◽  
Glycoprotein I ◽  
Multistep Process ◽  
Subendothelial Matrix ◽  
Platelet Aggregates ◽  
Multimeric Protein ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe formation of thrombi is a multistep process involving several components, including von Willebrand factor (VWF). VWF is an adhesive multimeric protein, which acts as a molecular bridge between the subendothelial matrix and the glycoprotein Ib/IX/V receptor complex. Furthermore, VWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin αIIbβ3. In terms of thrombus formation, it is essential that VWF-platelet interactions occur timely, that is: it should happen not too early or too late. Given the co-existence of VWF and platelets in the circulation, this implies that there must be regulatory mechanisms that prevent premature formation of VWF-rich platelet aggregates that could occlude the vasculature. Indeed, several mechanisms have been identified at the level of VWF, which are dedicated to the prevention of excessive VWF-platelet interactions following endothelial release of VWF (which may include limited exposure to shear stress, the presence of Mg2+ ions, inhibition of VWF-platelet interactions by endothelial proteins, ADAMTS13-mediated proteolysis) and of circulating VWF-platelet aggregates during normal circulation (shielding of the platelet-binding A1 domain by other regions of the VWF molecule, inhibition of VWF-platelet interactions by β2-glycoprotein I). In the present review an overview of these mechanisms will be discussed.

Adhesive Domains in the Collagen III Fragment α1(III)CB4 that Support α2β1- and von Willebrand Factor-mediated Platelet Adhesion under Flow Conditions

1999 ◽  
Vol 82 (09) ◽  
pp. 1137-1144 ◽  
Author(s):  
Martin IJsseldijk ◽  
Glenda Heijnen-Snyder ◽  
Eric Huizinga ◽  
Laurence Morton ◽  
C. Graham Knight ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Recognition Site ◽  
Flow Conditions ◽  
Binding Studies ◽  
Collagen Iii ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySeven overlapping peptides derived from the bovine α1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin α2β1-recognition site has been assigned within this fragment to residues 522-528 of the collagen α1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both α2β1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solidphase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*-GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the α1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the α2 subunit of integrin α2β1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the α2β1-recognition site in this locality in α1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the α1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of α2β1-, vWF- and rLAPP-binding sites all in close proximity in α1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

scholarly journals A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2819-2828 ◽  
Author(s):  
Sara Zanardelli ◽  
Alain C. K. Chion ◽  
Evelyn Groot ◽  
Peter J. Lenting ◽  
Thomas A. J. McKinnon ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Surface Plasmon ◽  
Binding Assay ◽  
Initial Step ◽  
Static Conditions ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

AbstractADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD ∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

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 ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4363-4371 ◽  
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.

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