Von Willebrand Factor A1 Domain Research Articles

Echocardiographic Molecular Imaging of the Effect of Anticytokine Therapy for Atherosclerosis

Echocardiographic molecular imaging techniques are beginning to be applied to evaluate preclinical efficacy of new drugs. In a large clinical trial, anti-interleukin-1β (IL-1β) immunotherapy reduced atherosclerotic events, yet treatment effects were modest, and the mechanisms of action were not fully elucidated. We tested the hypothesis that echocardiographic molecular imaging can assess changes in vascular thromboinflammatory status in response to anti-IL-1β therapy. In wild-type and atherosclerotic mice deficient for the low-density lipoprotein-receptor and Apobec-1, closed-chest myocardial infarction (MI) was performed to mimic high-risk clinical cohorts. Control animals had sham surgery. Post-MI animals were randomized to either no therapy or anti-IL-1β immunotherapy, which was continued weekly. At post-MI day 3 or 21, invivo ultrasound molecular imaging of aortic VCAM-1, P-selectin, von Willebrand factor A1-domain, and platelet GPIbα in the thoracic aorta was performed. Aortic histology and NF-κB activity were assessed in atherosclerotic mice. In both atherosclerotic and wild-type mice, MI produced a several-fold increase (P<.05) in aortic molecular signals for P-selectin, VCAM-1, von Willebrand factor, and GPIbα. In atherosclerotic mice, signal remained elevated at day 21. Anti-IL-1β therapy completely abolished the post-MI increase in signal for all endothelial targets (P<.05 vs nontreated) at day 3 and 21. In atherosclerotic mice, MI triggered an increase in aortic plaque growth and macrophage content, a decrease in plaque collagen, and elevated aortic NF-κB (P<.05 for all changes). All of these remote plaque adverse changes were inhibited by anti-IL-1β therapy. Echocardiographic molecular imaging of the vascular endothelium can quantify the beneficial effects of therapies designed to suppress the proatherosclerotic arterial thromboinflammatory effects of alarmins such as IL-1β. This approach could potentially be used to evaluate the biologic variables that influence response in preclinical studies, and possibly to select patients most likely to benefit from therapy.

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.

Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D′D3 and D4 domains

AbstractWe observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D′D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient's daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B–like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

Von Willebrand Factor Predicts Mortality in ACS Patients Treated with Potent P2Y12 Antagonists and is Inhibited by Aptamer BT200 Ex Vivo.

von Willebrand factor (VWF) is crucial for arterial thrombosis and its plasma levels are increased in acute coronary syndromes (ACSs). The effects of conventional platelet inhibitors are compromised by elevated VWF under high shear rates. BT200 is a third-generation aptamer that binds and inhibits the A1 domain of human VWF. This article aims to study whether VWF is a predictor of mortality in ACS patients under potent P2Y12 blocker therapy and to examine the effects of a VWF inhibiting aptamer BT200 and its concentrations required to inhibit VWF in plasma samples of patients with ACS. VWF activity was measured in 320 patients with ACS, and concentration effect curves of BT200 were established in plasma pools containing different VWF concentrations. Median VWF activity in patients was 170% (interquartile range % confidence interval [CI]: 85-255) and 44% of patients had elevated (> 180%) VWF activity. Plasma levels of VWF activity predicted 1-year (hazard ratio [HR]: 2.68; 95% CI: 1.14-6.31; p < 0.024) and long-term (HR: 2.59; 95% CI: 1.10-6.09) mortality despite treatment with potent platelet inhibitors (dual-antiplatelet therapy with aspirin and prasugrel or ticagrelor). Although half-maximal concentrations were 0.1 to 0.2 µg/mL irrespective of baseline VWF levels, increasing concentrations (0.42-2.13 µg/mL) of BT200 were needed to lower VWF activity to < 20% of normal in plasma pools containing increasing VWF activity (p < 0.001). VWF is a predictor of all-cause mortality in ACS patients under contemporary potent P2Y12 inhibitor therapy. BT200 effectively inhibited VWF activity in a target concentration-dependent manner.

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.

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.

Binding of Factor VIII to D'D3 and A2 Domains of Von Willebrand Factor Facilitates Mechanical Unraveling of the A2 Domain

Background: Proteolytic cleavage of von Willebrand factor (VWF) by a plasma metalloprotease ADAMTS13 is critical for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (FVIII) through its light chain to VWF significantly accelerates the proteolytic cleavage of VWF by ADAMTS13 under fluidic shear. However, the mechanism underlying the FVIII-cofactor activity enhancing VWF proteolysis by ADAMTS13 under shear is not fully understood. Present study aims to test a hypothesis that FVIII binding to the D'D3 and/or other adjacent domains may facilitate a mechanic-induced conformational change in the central A2 domain of VWF, resulting in an increased accessibility and the cleavage of Tyr1605-Met1606 bond by ADAMTS13. Methods: Recombinant B-domainless FVIII and various VWF fragments (D'D3, D'D3A1, D'D3A1A2, and A2) with an Avi-His-tag on their N-termini and a Spy-tag on their C-termini were expressed and affinity-purified to homogeneity. Laser optical tweezers analysis determined the force-induced conformational changes of VWF fragments in the absence or presence of FVIII. Atomic force microscopy (AFM) and surface plasmon resonance (SPR) determined the binding events and kinetics between FVIII and various VWF domains. Results: Laser optical tweezers analysis demonstrated a Gaussian distribution of unfolding extension events upon pulling the single D'D3A1 fragment with the most probable extension distance of 22.8 ± 1.9 nm (mean ± SEM). Addition of 5 nM FVIII to the D'D3A1 or D'D3 fragment resulted in only a small decrease (~25%) or no change in the pulling extension profile. However, two peaks of Gaussian distribution were detected when pulling the D'D3A1A2 with one peak at ~18 nm and the other at ~40 nm. Addition of 5 nM FVIII to the D'D3A1A2 fragment, only one unfolding event was detected with the most probable unfolding extension distance of 24.5 ± 0.4 nm. Moreover, a single Gaussian distribution of unfolding extension profile was detected when pulling an isolated A2 fragment with the most probable unfolding extension of 31 ± 1 nm. Interestingly, addition of 5 nM FVIII resulted in a significant reduction of the most probable unfolding extension to 16.0 ± 0.4 nm, suggesting that FVIII may directly interact with A2 domain. AFM and SPR demonstrated the binding kinetics between FVIII and various VWF fragments. As shown, the intrinsic off-rate (k0) between FVIII and D'D3 was 0.19 ± 0.9 s-1 and the barrier width (Δx‡) between the naive state and the transition state was 1.5 ± 0.2 Å; the k0 between FVIII and D'D3A1A2 was 2.8 ± 1.0 s-1 and the barrier width (Δx‡) between the naive state and the transitional state was 1.7 ± 0.2 Å. Surprisingly, there was a detectable, but relatively weak binding between FVIII and A2 domain using AFM. SPR experiments confirmed the binding of FVIII to D'D3, D'D3A1A2, and A2 with the dissociation constant (KD) of ~2.9 nM, ~5.2 nM and ~1.6 nM, respectively. These results demonstrate that the specific interaction between FVIII and D'D3 or A2 domain. Conclusions: We have demonstrated for the first time using single-molecule force spectroscopy that binding of FVIII to the D'D3 and/or A2 domain of VWF may facilitate the force-induced conformational changes of the A2 domain, resulting in the enhanced proteolysis by ADAMTS13 under force. Our findings shed new light on how FVIII may regulate VWF proteolysis and function under (patho) physiological conditions. Disclosures Zheng: Shire/Takeda: Research Funding; Alexion: Speakers Bureau; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau.

Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα–adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function.

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2β1 integrin. Neither the α2β1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

Abstract 1553: Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies

Abstract Cancer immunotherapy with immune checkpoint inhibitors (CPI), interleukin (IL)-2, and IL-12 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-CTLA4 (αCTLA4) + anti-PD-L1 (αPD-L1) and the cytokines IL-2 and IL-12 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokines) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, with 57% of total injected dose depositing in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2/IL-12. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. CBD-fusion to IL-12 significantly decreased hepatotoxicity and systemic IFNg concentration. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. CBD-IL-12 therapy induced remission of B16F10 melanoma and EMT6 breast cancer, along with increased intratumoral IFNg concentration compared to normal IL-12 therapy. Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokines) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. Unlike other active tumor-targeting approaches, CBD-based targeting is novel in a sense that it exploits abnormal tumor vessel structure; this characteristic affords binding to the ubiquitous extracellular matrix protein in the tumor, while limiting exposure to other tissues. CBD-immunotherapeutics may efficiently activate tumor-antigen-specific T cells and increase intratumoral downstream cytokines while maintaining systemic immune homeostasis by avoiding influencing non-tumor antigen specific T cells and systemic cytokine release. Citation Format: Jun Ishihara, Ako Ishihara, Aslan Mansurov, Koichi Sasaki, Steve S. Lee, John-Michael Williford, Lambert Potin, Peyman Hosseinchi, Laura T. Gray, Kiyomitsu Katsumata, Stephen J. Kron, Melody A. Swartz, Jeffrey A. Hubbell. Collagen affinity improves safety and efficacy of antibody and cytokine cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1553.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing

AbstractDuring wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

Essentials Platelet adhesion to von Willebrand factor (VWF) is critical for hemostasis and thrombosis. Whether VWF can undergo phosphorylation is unknown. Family with sequence similarity 20 kinase phosphorylates VWF A2 domain at S1517 and S1613. Phosphorylation of VWF and VWF A1A2A3 domain at S1613 enhances platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) mediates platelet adhesion and contributes to hemostasis at sites of vascular injury as well as to arterial thrombosis. The A1A2A3 domains of VWF contain important sites that differentially participate in supporting platelet adhesion. FAM20c (family with sequence similarity 20, member C) has emerged as a serine/threonine kinase, which phosphorylates extracellular proteins containing the S-X-E/pS motifs that are also found within the VWF A domains. This is of interest because we and others have shown that structural modifications within these A domains influence the ability of VWF to support platelet adhesion. Objective We assessed if VWF A domains can be phosphorylated and the functional consequence of phosphorylated VWF. Results Here, we show that FAM20c phosphorylated purified plasma VWF, VWF A1A2A3 protein, isolated A2 domain, but not A1 and A3 domain proteins, invitro. FAM20c phosphorylated the isolated A2 domain at S1517 and S1613 within the S-X-E recognition motif, with S1613 being the major phosphorylation site. Mass spectrometry analysis of purified plasma VWF from healthy donors revealed several phosphorylation sites, including the S1613 in the A2 domain. VWF A1A2A3 domain protein phosphorylated at S1613 promoted stable platelet adhesion and microthrombi at high shear stress. Lastly, under high shear stress VWF treated with FAM20c and ATP robustly supported platelet adhesion, compared to VWF treated with FAM20c in the absence of ATP. Conclusion These outcomes indicate that VWF can be phosphorylated by FAM20c invitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

Collagen affinity improves efficacy and safety of antibody and cytokine cancer immunotherapy.

e14232 Background: Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 have demonstrated clinical antitumor efficacy but is frequently accompanied with severe side-effects caused by excessive and systemic immune system activation. Methods: We addressed this need by targeting both the CPI antibodies anti-CTLA4 (aCTLA4) + anti-PD-L1 (aPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain. This approach harnesses the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. Results: We show that intravenously (i.v.) administered CBD protein accumulated in tumors in an orthotopic MMTV-PyMT breast tumor model. CBD conjugation or fusion decreased the systemic toxicity of both aCTLA4+aPD-L1 combination therapy and IL-2. CBD-conjugation to CPI abolished T cell infiltration into the liver and eliminated hepatotoxicity as well as decreased systemic cytokine release in the blood serum. CBD-fusion to IL-2 ameliorated capillary leak syndrome and pulmonary edema. These side-effects have been reported in the clinic. CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT slow growing breast cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. Conclusions: Our data suggest that the A3 domain of VWF can be used to engineer immunotherapies (e.g. CPI and cytokine) with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms.

Exploring the “minimal” structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering

AbstractHuman ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)–platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58–amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a “minimal” structure for allosterically regulated ADAMTS13.

Phylogenetic and functional analysis of ADAMTS13 identifies highly conserved domains essential for allosteric regulation

AbstractThe metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib

AbstractThe third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.