Von Willebrand Factor A1 Research Articles

The effects of shear stress on the interaction between ADAMTS13 and VWF

<p indent="0mm">Both ADAMTS13 (a disintegrin and metalloprotease with a thrombosspondin type-1 motif, member 13) and VWF (von Willebrand factor) are critical in the regulation of platelet adhesion in hemostasis and thrombosis. Dysregulation of their functions is associated with thrombotic thrombocytopenic purpura (TTP) and von Willebrand disease (VWD). VWF is a multimeric glycoprotein discovered by Erik A. von Willebrand in 1926. It can bind and stabilize coagulation factor VIII. In addition, it can mediate platelet adhesion by binding to the components of the blood vessel wall and the glycoprotein Ib (GPIb) on the platelet membrane. Under physiology, VWF is synthesized as a disulfide-linked dimer in the endoplasmic reticulum and transported into the Golgi apparatus for further processing. In the Golgi, the intra- and inter-VWF disulfide bonds are reorganized so that VWF can polymerize into macro-molecular helices and be packed into Weibel-Palade Bodies in a tubular form. Upon stimulation by blood vessel injury, packed VWF can be rapidly unwound and released into the blood vessels, forming millimeter-long protein fibers. Under the stretch of increased shear force at the injury site, the cryptic platelet binding site on the A1 domain of VWF will be exposed, enabling the recruitment and aggregation of platelets on VWF fibers for hemostasis. However, the accumulation of ultra-long VWF in the blood would lead to the formation of microthrombi, blocking blood vessels and capillaries and causing the failure of multiple organs. To prevent thrombosis, multimeric VWF is cleaved by ADAMTS13 into short fragments to reduce platelet binding and aggregation. As such, the length and distribution of VWF multimers, which ADAMTS13 regulates, are essential for the precise balance between hemostasis and thrombosis. Unlike other proteases, which are secreted in an inactive state and activated proteolytically, ADAMTS13 is secreted in an active form and circulates in the blood as an active enzyme. Its activity is regulated by VWF binding and shear stress. Under resting state, the head and the tail of ADAMTS13 interact with each other to mask the exosites of ADAMTS13 on its DTCS domains. VWF binding induces conformational changes of ADAMTS13 to expose its exosites. On the other hand, shear stress not only causes the conformational changes of VWF to expose its ADAMTS13 cleavage site on the VWF A2 domain, but also regulates the interactions between VWF and ADAMTS13. Studies have shown that, under shear stress, VWF and ADAMTS13 need to be properly aligned so that the VWF A2 domain can be positioned in the vicinity of the ADATS13 M domain for cleavage. In this article, we summarize recent progress in the structural and functional studies of VWF and ADAMTS13. Primarily, we focus on how the conformations and interactions between VWF and ADAMTS13 are regulated under static and shear force conditions. The collected information will be helpful for better understanding the regulation of ADAMTS13 and VWF activities under physiological and pathological conditions. Such understanding could facilitate the screening and development of novel therapeutics in the treatment of TTP and VWD.

Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and—at least bitiscetin-1—induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein—bitiscetin-3—were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit—which differs from that of both bitiscetin-3 subunits—the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear.

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

Development of a novel aptamer blocking the interaction between the VWF A1 domain and platelet GP Ib for the treatment of arterial thrombosis

A DNA aptamer to the von Willebrand factor (VWF) A1 domain, TAGX-0004, inhibits the binding of VWF to platelets. Nucleic acid aptamers are single-stranded DNA or RNA molecule forming three-dimensional structures that are capable of specifically binding to proteins and are expected to contribute to alternative medicine. ARC1779, an aptamer targeting the VWF A1 domain, had been evaluated in a phase II clinical trial of patients with acquired thrombotic thrombocytopenic purpura (aTTP); however, its development was terminated. Caplacizumab, an anti-VWF A1 domain nanobody, is now increasingly employed as first-line therapy for the treatment of aTTP in Western countries. However, there have been reports regarding adverse bleeding events and the high cost of the treatment. In this study, the inhibitory effects of TAGX-0004 were compared with those of ARC1779 and caplacizumab on in vitro platelet aggregation and thrombus formation. TAGX-0004 had an excellent high affinity to the VWF A1 domain and superior efficacy, such as its potent inhibitory activity in in vitro platelet aggregation and thrombus formation. Therefore, it can potentially overcome the problems associated with caplacizumab and can be developed as a promising drug not only for aTTP treatment but also for the treatment of the various VWF-mediated thrombotic disorders.

The soluble N-terminal autoinhibitory module of the A1 domain in von Willebrand factor partially suppresses its catch bond with glycoprotein Ibα in a sandwich complex.

von Willebrand factor (VWF) senses and responds to the hemodynamic forces to interact with the circulatory system and platelets in hemostasis and thrombosis. The dark side of this mechanobiology is implicated in atherothrombosis, stroke, and, more recently, the COVID-19 thrombotic symptoms. The force-responsive element controlling VWF activation predominantly resides in the N terminal auto-inhibitory module (N-AIM) flanking its A1 domain. Nevertheless, the detailed mechano-chemistry of soluble VWF N-AIM is poorly understood at the sub-molecular level as it is assumed to be unstructured loops. Using the free molecular dynamics (MD) simulations, we first predicted a hairpin-like structure of the soluble A1 N-AIM derived polypeptide (Lp; sequences Q1238-E1260). Then we combined molecular docking and steered molecular dynamics (SMD) simulations to examine how Lp regulates the A1-GPIbα interaction under tensile forces. Our simulation results indicate that Lp suppresses the catch bond in a sandwich complex of A1-Lp-GPIbα yet contributes an additional catch-bond residue D1249. To experimentally benchmark the binding kinetics for A1-GPIbα in the absence or presence of Lp, we conducted the force spectroscopy-biomembrane force probe (BFP) assays. We found similar suppression on the A1-GPIbα catch bond with soluble Lp in presence. Clinically, as more and more therapeutic candidates targeting the A1-GPIbα axis have entered clinical trials to treat patients with TTP and acute coronary syndrome, our work represents an endeavor further towards an effective anti-thrombotic approach without severe bleeding side effects as most existing drugs suffer.

Phage Display Functionally Defines Variants in the Von Willebrand Factor Platelet Binding Domain

Missense variants in the von Willebrand factor (VWF) platelet binding domain, A1, may pathologically hyperactivate or weaken interactions with its platelet receptor, GPIbα, and lead to von Willebrand disease (VWD) subtypes 2B or 2M, respectively. Variants identified in VWD patients and tested as recombinant VWF have supported genotype-phenotype associations and subtyping of VWD by genetic analyses. However, novel variants, most classified as variants of uncertain significance (VUS) are poorly defined. To functionally characterize a large subset of VWF A1 variants (P1254-L1460), we screened a phage display library for binding to a recombinant form of GPIbα used to clinically assess VWF platelet binding activity, GPIbM. Comprised of ~5x10 6 independent clones, the phage display library contained 1,427 unique, missense variants (~36% of all possible single amino acid substitutions) which could be scored for significant enrichment, depletion, or no change following selection for GPIbM binding. The enrichment of phage displayed VWD variants previously classified as VWD subtype 2B significantly segregated from reported 2M variants (mean fold change from preselected phage ~1.06 for 2B vs. ~0.68 for 2M, p < 0.005). To further validate these findings, five depleted, four unchanged, and seven enriched VWF A1 variants were introduced into the full length VWF sequence by site-directed mutagenesis and expressed by transient transfection of HEK293T cells. Conditioned media were collected and analyzed for VWF level (VWF:Ag) and activity (VWF:GPIbM). Of the sixteen variants examined, fourteen (87.5%) exhibited a VWF GPIbM:Ag ratio that was concordant with the phage display findings. Furthermore, the VWF GPIbM:Ag ratios were well correlated with the degree of enrichment by phage display (Pearson R = 0.69, p< 0.01). Taken together, these findings demonstrate phage display as a high content approach to measure and functionally define the platelet-binding activity of genetic variants within the VWF A1 domain. DisclosuresGinsburg: Takeda: Patents & Royalties.

Primary Myeloma Cell Induced Von Willebrand Factor Release from the Endothelium Is Mediated By VEGF and Attenuated By Heparin

IntroductionMultiple Myeloma (MM) remains an incurable disease and is associated with high rates of venous thromboembolism (VTE), the biological basis for which is not fully understood. Critically, VTE is associated with increased mortality in MM. This highlights the clinical importance of understanding cancer-coagulation crosstalk in MM. Accumulating evidence demonstrates that many solid tumours trigger endothelial cell (EC) activation with resultant von Willebrand Factor (VWF) secretion proposed to contribute to both risk of VTE and cancer metastasis. However, the interplay between VWF and MM disease biology remains poorly defined.Methods/Results100 patients with plasma cell disorders were recruited to this study. Significantly elevated plasma VWF antigen (VWF:Ag) levels were observed in patients with newly diagnosed MM (NDMM) compared to those with monoclonal gammopathy of undetermined significance (MGUS) or smouldering MM (SMM) (median 292.7 IU/dL vs 133.1 IU/dL; P<0.0001). Moreover, VWF:Ag levels in those with relapsed/refractory MM (RRMM) were further elevated (median 339 IU/dL; P<0.0001]). VWF:Ag levels also appear to correlate with disease response to therapy, with lower levels seen in patients achieving a complete response/very good partial response compared with those gaining only a partial response or worse (median 154.45 IU/dL vs 338.5 IU/dL; P<0.05).We also observed increased VWF collagen binding activity in NDMM/RRMM compared with MGUS/SMM (median 455.7 IU/dL vs 189.7 IU/dL; P<0.05). This qualitative measurement is sensitive to alterations in haemostatically active high molecular weight multimers of VWF secreted upon EC activation. Supporting this, raised plasma VWF propeptide (VWFpp) levels, a marker of acute EC activation, were seen in those with NDMM/RRMM compared with MGUS/SMM (median 182 IU/dL vs 133.01 IU/dL; P<0.05). Reduced VWFpp/VWF:Ag ratios were seen in those with NDMM/RRMM, indicating decreased circulatory clearance of VWF. We hypothesise that MM may exhibit a ‘double-hit’ in regulation of plasma VWF:Ag with increased secretion of VWF but also simultaneously reduced VWF clearance leading to sustained and markedly elevated systemic plasma VWF levels.Next we measured VWF:Ag levels in bone marrow (BM) samples from 10 patients and found that VWF is released locally within the BM niche (median 190.2 IU/dL). Co-culture of primary human ECs with supernatant from freshly isolated primary MM cells or several Human Myeloma Cell Lines (HMCLs) demonstrated that MM cells stimulate release of VWF from ECs in a rapid manner, suggestive of Weibel-Palade body exocytosis (median 34.9ng/ml vs 9.2ng/ml in untreated EC; P<0.05). Elevated levels of Vascular Endothelial Growth Factor-A (VEGF-A), a potent EC activator, were observed in MM cell supernatants. Pre-treatment of primary MM cells or HMCLs with anti-VEGF antibody bevacizumab abolished EC VWF secretion in a dose dependant manner (32.3ng/ml vs anti-VEGF treated 10.1ng/ml P<0.0001). Furthermore, clinically relevant doses of low molecular weight heparin (LMWH) also attenuated VWF secretion (29.6ng/ml vs LMWH treated 15ng/ml; P<0.05). Measurement of VEGF-A in primary MM cell and HMCL supernatant treated with LMWH or bevacizumab confirmed significant reductions in VEGF-A levels. Together these data indicate that stimulation of VWF release from the endothelium by MM cells is mediated by VEGF-A and directly inhibited by LMWH treatment.Using flow cytometry, we assessed the direct interaction of MM cells and VWF in vitro. Both primary MM cells and HMCLs bound significantly to human recombinant VWF in a dose-dependent manner. Binding was reduced by 50% following treatment with LMWH (P<0.05). Given that the VWF A1 domain contains a heparin binding motif, we speculate that MM cell adhesion to VWF may be at least in part mediated by this domain and future work focuses on elucidating the receptors involved.ConclusionThese data suggest that MM is not only associated with a marked quantitative increase in plasma VWF:Ag, but also impacts VWF functional activity. Our novel data help define the biological mechanisms underpinning elevated VWF levels in MM, with a key contribution of VEGF-A secreted directly from MM cells in the BM microenvironment. Collectively, our findings provide insights into cancer-coagulation crosstalk in MM and may help identify novel therapeutic targets to reduce VTE risk and disease progression. DisclosuresGlavey: Celgene and BMS company: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Quinn: Takeda: Honoraria. O'Sullivan: Leo Pharma: Research Funding.

Fibronectin binding to von Willebrand factor occurs via the A1 domain

BackgroundCollagen interactions with von Willebrand factor (VWF) perform an important role in initiation of hemostasis. ObjectivesWe hypothesized that in addition to collagen, other extracellular matrix (ECM) proteins such as fibronectin can bind VWF. MethodsFibronectin‐VWF interactions were measured by ELISA using both plasma‐derived and recombinant VWF–containing variants in specific domains. Inhibition was measured by antibody competition using antibodies directed against both VWF and fibronectin. Binding affinities were measured by the Octet Biosensor for fibronectin and collagen IV. ResultsFibronectin was able to bind both plasma‐derived and recombinant wild‐type VWF. This interaction was inhibited by both anti‐VWF antibodies and collagen types III and IV. Several VWF A1 domain variants in the region of the collagen IV binding site also demonstrated absent fibronectin binding, as did variants with defects in high‐molecular‐weight multimers. Binding affinity testing showed fibronectin has a strong affinity for VWF, in a range similar to that of collagen IV. Fibronectin binds VWF via a restricted region of the A1 domain. This interaction requires high‐molecular‐weight multimers and is similar to that seen with vascular collagens. ConclusionsTherefore, VWF would appear to be the common factor linking platelet adhesion to various ECM proteins and facilitating hemostasis under conditions of ECM exposure. [Display omitted]

Mapping the Masked Region of the A1 Domain in Full-Length VWF

Von Willebrand factor (VWF) is the largest protein in plasma and promotes the early hemostatic response following vascular injury. The hemostatic response of VWF relies on the exposure of its A1 domain, which recognizes its receptor GPIbα on the platelet surface. To avoid binding and activating platelets in the absence of injury, the A1 domain is dormant in full-length VWF through a mechanism that is not completely understood. We recently characterized two recombinant A1 fragments (longA1, residues 1238-1472; and shortA1, residues 1261-1472) with differential GPIbα-binding affinities by hydrogen-deuterium exchange (HDX) mass spectrometry. We found that N- and C-terminal residues flanking the A1 domain (residues 1238-1271 and 1459-1472) cooperatively constitute an autoinhibitory module (AIM) that masks a region in A1 and impedes its binding to GPIbα[1]. To determine if the A1 domain in full-length VWF is “masked” in the same manner, we have characterized human plasma-derived VWF using the same HDX approach.On-column peptic fragmentation was performed following HDX from 10 to 10,000 sec. We obtained 946 sequenced peptides covering 96.4% of the VWF sequence with an average redundancy of 6 per residue. Among them, 113 peptides covered the A1 domain, allowing HDX analysis of A1 at high resolution. We found that residues around the 1272-1458 disulfide bond, helix α1, loops α1β2 and β3α2 in the A1 domain, which are protected by the AIM in the longA1 fragment (residues 1238-1472), are deeply protected in full-length VWF, suggesting that AIM in full-length VWF likely masks A1 in a similar pattern. Additionally, sheet β3, helix α3, and loops β4α3 in the A1 domain of full-length VWF reported lower HDX rates than their counterparts in the shortA1 fragment. These regions, along with those protected by AIM, form a contiguous surface that significantly overlaps with the GPIbα-binding interface (Fig. 1). Residues in loop β6α6, helix α6 and helix α1 also reported lower HDX rates, but they are separate from the regions protected by AIM and the GPIbα-binding interface.In summary, our results suggest that AIM and likely other domains in full-length VWF are in direct contact with the A1 domain and regulate the hemostatic properties of VWF.Figure 1: Difference in HDX between isolated VWF A1 domains and full length VWF.1 Deng W, Wang Y, Druzak SA, Healey JF, Syed AK, Lollar P, Li R. A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor. Journal of thrombosis and haemostasis : JTH . 2017. 10.1111/jth.13775. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand Factor A1 Domain Binds to Clusters II and IV of Low-Density Lipoprotein Receptor-Related Protein 1

Introduction. Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein involved in hemostasis and thrombosis. Genetic and in vivo studies have identified low-density lipoprotein receptor-related protein 1 (LRP) as the major clearance receptor of VWF. The ligand-binding moiety of LRP is represented by complement-type repeats grouped into four clusters, among which clusters II and IV are prominent in ligand binding. In in vitro studies under shear stress conditions, VWF acquires an unfolded conformation and interacts with LRP. More than 30 mutations of VWF have been reported to associate with its increased plasma clearance and result in type 2B von Willebrand disease. The majority of these mutations are located within the VWF A1 domain (A1), which is in agreement with previous reports that isolated A1 binds to LRP. However, the interactive site of LRP has not been identified.Objective. We aim to characterize the LRP binding site for VWF by investigating the interaction of individual LRP clusters II-IV to different A1 variants.Experimental Approach. An A1-containing large N-terminal homo-dimeric proteolytic fragment of VWF (SPIII) was produced upon its proteolytic digestion by V8-protease. Alternatively, we generated three variants of recombinant A1: (i) A1(residues 1261-1478), (ii) a VWD type 2B mutant, A1(V1316M) (residues 1261-1478) and (iii) an N-terminus truncated form, (residues 1269-1478). These proteins were tested for binding to immobilized LRP, purified from human placenta, and recombinant LRP clusters II and IV by surface plasmon resonance.Results. We did not find any interaction between the SPIII proteolytic fragment and LRP, suggesting that this fragment may still preserve a folded conformation of VWF that masked its LRP-binding site. In turn, all three forms of recombinant A1 were found to bind LRP and its clusters II and IV. Notably, A1(V1316M) and A1(1269-1478) had higher affinity for LRP and its clusters than A1. This result is consistent with the enhanced clearance of the VWF(V1316M) in vivo and better exposure of the A1 site in a truncated form of A1 to the platelet receptor GpIb-alpha reported previously.Conclusions. Our data demonstrate that LRP has at least two sites for binding to VWF, which are located within clusters II and IV. This finding is in accordance with previous observations that LRP clusters II and IV can serve as “functional duplicates” for binding selected ligands. DisclosuresNo relevant conflicts of interest to declare.

Contrast-enhanced ultrasound molecular imaging of activated platelets in the progression of atherosclerosis using microbubbles bearing the von Willebrand factor A1 domain.

Platelet-endothelial interactions have been linked to increased inflammatory activation and a prothrombotic state in atherosclerosis. The interaction between von Willebrand factor (vWF)-A1 domain and platelet glycoprotein (GP) Ib/IX plays a significant role in mediating the adhesion of platelets to the injured endothelium. In the present study, contrast-enhanced ultrasound (CEU) molecular imaging with microbubbles bearing the vWF-A1 domain was performed to non-invasively monitor activated platelets on the vascular endothelium in the procession of atherosclerosis. A targeted CEU contrast agent was prepared by attaching the vWF-A1 domain to the shell of microbubbles (MbA1). Rat isotype control antibody was used to produce control (Mbctrl) microbubbles. The binding of MbA1 and Mbctrl to activated platelets was assessed in in vitro flow chamber experiments. Apolipoprotein E (ApoE-/-) deficient mice were studied as a model of atherosclerosis. At 8, 16 and 32 weeks of age, CEU molecular imaging of the proximal aorta with MbA1 and Mbctrl was performed and the imaging signals from microbubbles were quantified. Atherosclerotic lesion severity and platelets on the endothelial surface were assessed by histology and immunohistochemistry. In in vitro flow chamber studies, attachment of MbA1 to activated platelets on culture dishes was significantly greater than that of Mbctrl across a range of shear stresses (P<0.05). The attachment of Mbctrl was sparse and not related to the aggregated platelets. As lesion development progressed in the ApoE-/- mice, molecular imaging of activated platelets demonstrated selective signal enhancement of MbA1 (P<0.05 vs. Mbctrl) at all ages. Selective signal enhancement from MbA1 increased from 8 to 32 weeks of age. Immunohistochemistry for GPIIb revealed the presence of platelets on the endothelial cell surface in each group of ApoE-/- mice and that the degree of platelet deposits was age-dependent. The results of the present study indicated that non-invasive CEU molecular imaging with targeted microbubbles bearing the vWF-A1 domain could not only detect activated platelets on the vascular endothelium but also indicate lesion severity in atherosclerosis.

Binding of Gpibα to VWF A2 Domain Alters Mechanical Unraveling of the A2 Domain

Background: Von Willebrand factor (VWF), is a large, multimeric plasma glycoprotein that plays a critical role in hemostasis. VWF is synthesized and secreted as ultra large (UL) multimers that contain up to 100 protomers. If not processed by ADAMTS13, a plasma metalloprotease, ULVWF may initiate the spontaneous formation of life-threatening thrombi, as seen in thrombotic thrombocytopenic purpura (TTP). The cleavage site is buried under the central β-sheet within the VWF-A2 domain and tensile force is required to expose the cleavage site for ADAMTS13. Our prior studies demonstrate that several VWF-binding proteins, including coagulation factor VIII, apoB100/LDL, as well as the ectodomains of platelet glycoprotein Iba (GPIba), appear to function as cofactors that facilitate the proteolytic cleavage of VWF by ADAMTS13 under shear. However, the mechanism underlying GPIba enhancing effect on ADAMTS13-mediated VWF proteolysis is yet to be determined. Methods: Recombinant human GPIbα was purchased from Sino Biological. The recombinant VWF-A2 domain fragment with a SpyTag on the N-terminus and an AviTag-HisTag on the C-terminus was expressed in the HEK 293T cells and affinity-purified by the Ni-NTA affinity chromatography. Biotinylation was performed in vitro using a biotin-labeling kit. The binding between VWF-A2 and GPIbα was studied by a custom-built atomic force microscope (AFM) using our established single-molecule binding study protocol. MicroScale Thermophoresis was conducted by a Monolith device to detect the binding affinity between the VWF-A2 and GPIbα. A dual-beam mini-tweezers instrument was utilized to characterize the force-induced conformational changes of the A2 domain in the absence and presence of GPIbα with a pulling speed of 200 nm/s. Results: AFM results indicated that specific binding interactions occurred between GPIbα and VWF-A2. Monolith MST assay revealed a strong binding affinity (Kd of ~20 nM) between GPIbα and the VWF-A2 fragment. In the optical tweezer study, pulling on a single VWF-A2 resulted in an unfolding event at 10-30 pN with an extension ranging from 30 to 40 nm (Fig. 1A). Gaussian fits of the unfolding extension distributions revealed a most probable force-induced extension of 34.87 ± 2.2 nm (mean ± SEM) (Fig. 1B). Addition of 100 nM of GPIbα led to a noticeable decrease in both unfolding force and extension of VWF-A2 (Fig. 1A). The most probable unfolding extension reduced to 16.05 ± 0.2 nm in the presence of 100 nM of GPIbα (Fig. 1B), indicating the binding of GPIbα may influence mechanical unfolding of VWF-A2. Further, the unfolding results were analyzed by a worm-like chain model fit, which yielded a contour length for the initially folded structure of VWF-A2 at 58.83 ± 2.0 nm (mean ± SEM) and 24.53 ± 0.2 nm in the absence and presence of GPIbα, respectively (Fig. 1C), indicating that the specific interactions between GPIbα and A2 domain may partially unfold the A2 domain. Conclusions: These results demonstrate for the first time that binding of GPIbα to VWF-A2 may alter the force-induced conformational changes in the A2 domain. Under physiological conditions, the glycocalicin (or soluble GP1bα) may bind the VWF-A2 and cause A2 partial unfolding, which may result in excessive cleavage of VWF by ADAMTS13, thus regulating hemostasis. Figure legend: Fig.1. GPIbα influences the mechanical unfolding of the A2 domain of VWF. (A) Typical optical tweezer pulling traces of A2 domain in the absence (red) and presence (blue) of GPIbα (100 nM). The arrows point to the unfolding events. The pulling speed is 200 nm/s. (B) The histograms of the unfolding extension of pulling VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα at 200 nm/s. Solid lines are Gaussian fits to the distributions. (C) The relationship between unfolding force (pN) and unfolding extension (nm) of pulling the VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα. The data are fitted to the worm-like chain model (solid lines). Horizontal and vertical error bars are one standard deviation for force and half width of the half bin width for extension, respectively. Figure Disclosures Cao: Ivygen: Consultancy; Bayer: Research Funding. Zheng:Sanofi: Consultancy, Speakers Bureau; Clotsolution: Other: Co-Founder; Alexion: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Von Willebrand factor promotes platelet-induced metastasis of osteosarcoma through activation of the VWF-GPIb axis

von Willebrand factor (VWF) is exclusively expressed in endothelial cells (ECs) and megakaryocytes, which plays a crucial role in the initiation of arterial thrombosis. Recent studies have shown that VWF is also expressed in osteosarcoma (OS) cells and participates in adhesion of cancer cells to platelets, thus promoting metastasis of OS cells. However, it is unclear how OS cell-derived VWF-platelet interaction contributes to the metastasis of OS. We hypothesized that the interaction is mediated by the binding between VWF A1 and GPIbα of platelets, a molecular mechanism similar to that of thrombosis. The increased expression of VWF in SAOS2 cells may contribute to the enhancement of platelet adhesion through the VWF-GPIb pathway, which could promote the migration and invasion capacities of SAOS2 cells in vitro. Antibodies that block the pathway could significantly inhibit the platelet-induced metastasis of OS cells. Our results suggest a theoretical basis for the development of new anti-OS metastasis drugs, and further enrich the mechanism of OS metastasis.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

The development and characterization of a long acting anti‐thrombotic von Willebrand factor (VWF) aptamer

BackgroundThrombus formation involves coagulation proteins and platelets. The latter, referred to as platelet‐mediated thrombogenesis, is predominant in arterial circulation. Platelet thrombogenesis follows vascular injury when extracellular von Willebrand factor (VWF) binds via its A3 domain to exposed collagen, and the free VWF A1 domain binds to platelet glycoprotein Ib (GPIb). ObjectivesTo characterize the antiplatelet/antithrombotic activity of the pegylated VWF antagonist aptamer BT200 and identify the aptamer VWF binding site. MethodsBT100 is an optimized aptamer synthesized by solid‐phase chemistry and pegylated (BT200) by standard conjugation chemistry. The affinity of BT200 for purified human VWF was evaluated as was VWF inhibition in monkey and human plasma. Efficacy of BT200 was assessed in the monkey FeCl3 femoral artery thrombosis model. ResultsBT200 bound human VWF at an EC50 of 5.0 nmol/L and inhibited VWF A1 domain activity in monkey and human plasma with mean IC50 values of 183 and 70 nmol/L. BT200 administration to cynomolgus monkeys caused a time‐dependent and dose‐dependent effect on VWF A1 domain activity and inhibited platelet function as measured by collagen adenosine diphosphate closure time in the platelet function analyzer. BT200 demonstrated a bioavailability of ≥77% and exhibited a half‐life of >100 hours after subcutaneous injection. The treatment effectively prevented arterial occlusion in an FeCl3‐induced thrombosis model in monkeys. ConclusionsBT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non‐human primates. These data including a long half‐life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.

The von Willebrand Factor A1 domain mediates thromboinflammation, aggravating ischemic stroke outcome in mice.

Von Willebrand factor (VWF) plays an important role in ischemic stroke. However, the exact mechanism by which VWF mediates progression of ischemic stroke brain damage is not completely understood. Using flow cytometric analysis of single cell suspensions prepared from brain tissue and immunohistochemistry, we investigated the potential inflammatory mechanisms by which VWF contributes to ischemic stroke brain damage in a mouse model of cerebral ischemia/reperfusion injury. Twenty-four hours after stroke, flow cytometric analysis of brain tissue revealed that overall white blood cell recruitment in the ipsilesional brain hemisphere of VWF knockout mice was two times lower than that in wildtype mice. More detailed analysis showed a specific reduction of proinflammatory monocytes, neutrophils and T cells in the ischemic brain of VWF knockout mice compared to wild-type mice. Interestingly, histological analysis revealed a substantial number of neutrophils and T cells still within the microcirculation of the stroke brain, potentially contributing to the noreflow phenomenon. Specific therapeutic targeting of the VWF A1 domain in the wild-type mice resulted in reduced numbers of immune cells in the affected brain and protected mice from ischemic stroke brain damage. More specifically, recruitment of proinflammatory monocytes was reduced two-fold, neutrophil recruitment was reduced five-fold and T-cell recruitment was reduced two-fold in mice treated with a VWF A1-targeting nanobody compared to the recruitment in mice receiving a control nanobody. In conclusion, our data identify a potential role for VWF in the recruitment of proinflammatory monocytes, neutrophils and T cells to the ischemic brain through a mechanism that is mediated by its A1 domain.

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

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 USA. 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 suggest that TAGX-0004 possesses better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

Injury Characteristics and von Willebrand Factor for the Prediction of Acute Respiratory Distress Syndrome in Patients With Burn Injury: Development and Internal Validation.

To derive and validate a prediction model for the development of ARDS in burn-injured patients. Burn injury carries the highest incidence of acute respiratory distress syndrome (ARDS) among all predisposing conditions, but few studies exist on risk factors in these patients. Studies employing biomarkers and clinical risk factors for predicting ARDS mortality have recently been examined but none exist for onset of ARDS nor in patients with burn injury. This was a prospective multicenter study of 113 patients with isolated burn injury or inhalation injury. Clinical variables and plasma biomarkers representative of endothelial injury, epithelial injury, or inflammation were collected within 24 hours of admission. The most parsimonious model was chosen by considering discrimination, calibration, and model fit. Among the biomarkers measured in patients with burn injuries, a one-standard deviation increase in log-transformed levels of the A2 domain of von Willebrand factor in the first 24 hours was most strongly associated with the development of ARDS (OR 7.72; 95% CI: 1.64-36.28, P = 0.03). Of candidate models, a 3-variable model with %TBSA, inhalation injury, and von Willebrand factor-A2 had comparable discrimination to more complex models (area under the curve: 0.90; 95% CI 0.85-0.96). The 3-variable model had good model fit by Hosmer-Lemeshow test (P = 0.74) and maintained similar discrimination after accounting for performance optimism (Bootstrapped area under the curve: 0.90; 95% CI: 0.84-0.95). The 3-variable model with %TBSA, inhalation injury, and von Willebrand factor could be used to better identify at-risk patients for both the study and prevention of ARDS in patients with burn injury.

ADAMTS13, an Anti-thrombotic Protein: Evidence Outside of Thrombotic Thrombocytopenic Purpura

von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis. VWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and the site of injured or activated blood vessels. Biosynthesis of VWF is restricted to endothelial cells and megakaryocytes. Endothelial VWF is constitutively secreted into plasma and subendothelium, or is stored as "ultra-large" (UL)-VWF multimers in endothelial Weibel-Palade bodies. VWF produced in megakaryocytes is packaged as UL-VWF in the a-granules of platelets. VWF stored in endothelial and platelet storage organelles is secreted in a regulated process in response to stimulation by secretagogues. Absence or dysfunction of VWF results in bleeding symptoms, as observed in patients with von Willebrand disease. An abnormally high activity of VWF can lead to thrombotic events. Interestingly, the activity of VWF is determined by the size of its multimers and UL-VWF can spontaneously form platelet aggregates. An important regulator of VWF size is the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which digests large thrombogenic VWF molecules into smaller, less reactive multimers via cleavage of the Y1605-M1606 bond in the VWF A2 domain. ADAMTS13 is mainly synthesized in the liver by hepatic stellate cells, but other sites of synthesis, including renal podocytes, tubular epithelial cell, platelets and endothelial cells, have also been described. ADAMTS13 is released as an active enzyme into the circulation with no physiological inhibitors. Reduced or absent ADAMTS13 activity causes the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), characterized by VWF and platelet-rich microthrombi that cause multiple organ failure and even death when left untreated. Besides its clear role in the pathophysiology of TTP, the anti-thrombotic and even anti-inflammatory properties of ADAMTS13 have also become apparent in various other thrombotic conditions. High VWF levels and low ADAMTS13 levels are associated with increased risk or even worse outcome of cardiovascular disease, including ischemic stroke and myocardial infarction. Preclinical studies in mouse models showed the beneficial effect of ADAMTS13 in both cerebral and myocardial ischemia/ reperfusion injury by decreasing both thrombosis and inflammation. In addition, ADAMTS13 was shown to also exert a direct thrombolytic effect on VWF-rich thrombi. In a mouse model of ischemic stroke, this thrombolytic activity resulted in efficient lysis of intracranial thrombi that were resistant to standard treatment with tissue plasminogen activator. Hence, ADAMTS13, as a therapeutic agent, could become an interesting avenue, not only to manage TTP, but also to treat other thrombotic complications. Disclosures De Meyer: Fonds voor Wetenschappelijk Onderzoek: Research Funding; KU Leuven: Employment, Research Funding; Ablynx: Consultancy, Research Funding; Cerenovus: Membership on an entity's Board of Directors or advisory committees; WhiteSwell: Consultancy.