Von Willebrand Factor A1 Domain Research Articles

Caplacizumab as an emerging treatment option for acquired thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare disease with a mortality rate of over 90% if left untreated. Therapeutic plasma exchange (PEX) is the mainstay of treatment of acquired TTP (aTTP), and with the introduction of PEX, the mortality rate declined dramatically below 20%. Although PEX together with corticosteroids are the backbone of the upfront management of patients with aTTP with successful outcomes, patients may remain refractory and/or relapse following an initial response to this treatment. There are some therapeutic options, which can be used among these patients, helping in improving outcomes of aTTP. Caplacizumab (formerly ALX-0081 or ALX-0681) is a humanized single-variable domain immunoglobulin that recognizes the human von Willebrand factor (vWF) A1 domain and inhibits the vWF–platelet glycoprotein 1b-alpha (GP1b-α) interaction. The drug was first developed for the prevention of thrombosis in high-risk patients with acute coronary syndrome undergoing percutaneous coronary intervention; however, drug development for this indication has been discontinued. Recently, caplacizumab received its first approval following Phase II TITAN and Phase III HERCULES trials in the European Union (EU) for the treatment of acute episode of aTTP in adult patients, in addition to PEX and immunosuppression. This review focuses on the use of caplacizumab as an emerging treatment option in patients with aTTP.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ‘‘active” conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4–7.2% and 4.1–9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1–424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6–154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

Defective collagen binding and increased bleeding in a murine model of von Willebrand disease affecting collagen IV binding

Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to typeIV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its invivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagensI and IV was measured. VWF-dependent platelet adhesion to collagenIV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagenIV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagenIV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagenIV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagenIV binding.

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

<Introduction>von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction with its A1 domain and platelet glycoprotein 1b. Thus, VWF A1 domain was thought to be a good therapeutic target candidate for VWF mediated thrombosis. In this study, we analyzed the inhibitory effects of a novel DNA aptamer (TAGX-0004) on platelet aggregation compared with another VWF A1 domain binding aptamer (ARC1779) which had entered to Phase II clinical trial for acquired thrombotic thrombocytopenic purpura (TTP) in 2011.<Methods>TAGX-0004 was generated by SELEX (systematic evolution of ligands by exponential enrichment) and consisted of artificial nucleic acid base, 7-(2-thienyl) imidazo [4,5-b] pyridine (Ds) as well as natural bases (Adenine, Thymine, Cytosine and Guanine). The dissociation constant (Kd) of the aptamers was analyzed by electrophoretic mobility shift assay (EMSA). 100 nM of each DNA aptamer was mixed with A1 domain of VWF protein (final concentration was 0 to 800 nM) in binding buffer (1x PBS, 0.005% NP-40) and incubated for 30 min at 37 °C. The samples were subjected to EMSA with 8% native PAGE, then stained by SYBR Gold. Kd value was determined with scatchard plot analysis. To characterize the binding sites of VWF A1 to these DNA aptamers, various mutants of VWF A1 domain were generated with an alanine substitution technique.Platelet aggregation was induced with ristocetin (RIPA), botrocetin (BIPA), collagen, epinephrine and adenosine diphosphate (ADP). The change of light transmission rate of platelet rich plasma (PRP) compared to platelet poor plasma (PPP) at 37 °C was recorded for 6 min, then the inhibition ratio of platelet aggregation was determined. Total thrombus-formation analysis system (T-TAS) (Fujimori Kogyo Co. Ltd., Tokyo, Japan), which is a novel micro-chip flow-chamber system, was employed to analyze thrombus formation visually and quantitatively in whole blood samples. The micro-chip coated with type 1 collagen was used for this analysis. Anti-VWF A1 inhibitory effects of these aptamers at high shear stress (initial rate of 2000 s-1) were calculated by continuous pressure levels within its narrow capillary. The course of thrombus formation was also optically recorded with a video-microscope located under the microchip.<Results>Biophysical interaction analysis using EMSA showed that TAGX-0004 had approximately 15-times higher binding activity to VWF A1 domain than ARC1779. Based on an alanine scan analysis, it was revealed that a couple of residues we tested were critical for binding of ARC1779 but not for TAGX-0004, which indicating that TAGX-0004 interacts with VWF A1 domain via different amino acid residue(s).Both aptamers did not inhibit the platelet aggregation induced by collagen, epinephrine or ADP. In RIPA, the 80% inhibition was observed by TAGX-0004 and ARC1779 at a final concentration of 10 nM and 750 nM, respectively. In BIPA, those were seen at those of 50 nM and 1000 nM, respectively. In T-TAS analysis, TAGX-0004 inhibited the thrombus formation completely at a final concentration of 100 nM, whereas ARC1779 exhibited partial inhibition of thrombosis formation even at 1000 nM. These results indicated that TAGX-0004 had stronger inhibitory effect on the platelet aggregation compared with ARC1779 under both static and high-shear conditions.<Conclusions>In the present study, we showed the affinity to VWF-A1 domain of TAGX-0004 was stronger than that of ARC1779. In addition, TAGX-0004 was more effective in suppressing platelet aggregation under both static and high-shear stress condition than ARC1779. In the published results of clinical study of ARC1779, there were no hemorrhagic complications in the small clinical trial of patients (7 patients of ARC1779 group) despite the almost complete suppression of VWF function in severely thrombocytopenic patients. Therefore, TAGX-0004 could be developed as a promising therapeutic agent for VWF mediated thrombotic disorders, such as acute coronary syndrome and TTP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Site-Directed Pegylation at Specific Sites Significantly Prolongs the Half-Life of A1A2A3-VWF By Markedly Attenuating LRP1-Mediated Clearance

IntroductionDeficiencies of both von Willebrand Factor (VWF) and FVIII are associated with significant bleeding phenotypes. Consequently, patients with VWD or hemophilia A commonly require replacement therapy with coagulation factor concentrates. However, as infused VWF and FVIII have relatively short plasma half-lives, patient therapy generally necessitates frequent re-dosing. Development of a long-acting rVWF therapy thus represents an important unmet clinical need. We and others have previously demonstrated that the A1A2A3 domains of VWF play a critical role in regulating macrophage-mediated clearance of VWF in vivo. Importantly, crystal structures of the A-domains have also well characterized. In this study, we sought to utilize this data to investigate the hypothesis that site-specific PEGylation within the A1A2A3 domains could be used as a novel strategy to inhibit macrophage-mediated clearance, and thereby inform development of a rVWF molecule with extended plasma half-life.MethodologySite-directed mutagenesis was used to engineer novel surface cysteine residues at selected sites within A1A2A3-VWF. Following purification and characterization, individual A1A2A3 cysteine variants were PEGylated using 40kDa PEG maleimide. Clearance of unPEGylated and PEGylated A1A2A3 variants were assessed in VWF-/- mice. VWF-macrophage interactions were quantified in vitro using differentiated THP-1 macrophages. VWF binding to LRP1 clearance receptor was assessed using both immunosorbant assays and Surface Plasmon Resonance.ResultsNovel single cysteine residues were introduced at stringently selected sites within A1A2A3-VWF. These sites spanned all 3 A-domains and included; S1286C, Q1353C, M1545C, L1591C, V1636C, Q1652C, V1803C and S1807C. Interestingly, the introduction of these novel cysteine residues in both the A1 and A3 domains of VWF did not alter the rate of VWF clearance compared to WT A1A2A3-VWF. Conversely however, the A2 domain was less tolerant for the insertion of cysteines, with L1591C and V1636C variants demonstrating a significantly reduced VWF plasma half-life of approx. 1.5 fold versus WT-A1A2A3 (p<0.05).Subsequently, the engineered cysteine residues were modified by covalent attachment of a 40kDa branched PEG molecule. All variants achieved greater than 80% PEG conjugation efficiency, except V1636C which was eliminated from further study. Remarkably, PEG conjugation displayed site-specific effects on the in vivo half-life of A1A2A3-VWF. For example, PEGylation at S1286C within the A1 domain resulted in a marked increased in VWF half-life compared to WT-A1A2A3 VWF (92.4±6 vs 18.3±0.9 mins, respectively, p<0.001). Conversely, PEGylation at the adjacent site in the A1 domain, Q1353C, or downstream at M1545C within A2 had no significant effect on VWF half-life (23.3±1 and 20.8±3 mins, respectively). Interestingly, despite the fact that no previous roles have been described for the A3 domain of VWF in regulating its clearance, we observed a significant extension in VWF half-life for PEGylated variants within the A3 domain, V1803C and S1807C, (93.3±9 mins and 58.0±5 mins, respectively, p<0.05).Macrophage LDL receptor related protein 1 (LRP1) has been implicated as key cellular mediator of VWF clearance in vivo. Interestingly, in keeping with the reduced clearance observed for PEGylated VWF variants S1286C, V1803C and S1807C, binding of these variants to clearance receptor LRP1 cluster II and IV was ablated. Conversely, PEGylated variants which failed to extend VWF half-life (Q1353C and M1545C) displayed LRP1 binding that was comparable to WT-A1A2A3 VWF. Interestingly, PEGylation at specific sites in A2 (L1591C and Q1652C) which served to increased VWF half-life displayed normal binding to LRP1 cluster IV. However, binding of these variants to LRP1 cluster II was reduced by 90% compared to WT-A1A2A3.ConclusionCollectively, our novel data demonstrate that cysteine-directed PEGylation at specific sites within the A1 (S1286C), A2 (L1591C, Q1652C) and A3 (V1803C and S1807C) domains of A1A2A3-VWF inhibits binding to macrophage clearance receptor LRP1 in vitro. Consequently, these PEGylated A1A2A3-VWF variants demonstrate an extended circulatory half-life in vivo compared to wild type A1A2A3-VWF. Taken together, these results support the use of site-specific PEGylation as a potential approach to develop long-acting full length rVWF molecules. DisclosuresCooke:Pfizer: Employment. Terraube:Pfizer: Employment. Cohen:Pfizer: Employment. Pittman:Pfizer: Employment. Cunningham:Pfizer: Employment. Lambert:Pfizer: Employment. O'Donnell:Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; CSL Behring: Consultancy; Octapharma: Speakers Bureau; Leo Pharma: Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Bayer: Research Funding, Speakers Bureau; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.

Von Willebrand Factor Binding to Myosin Assists in Coagulation

Introduction: Von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factors V and X (Deguchi et al, Blood 2016;128:1807-1878). We hypothesized that VWF could also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation.Methods: Recombinant wild-type (WT) was transfected into HEK293T cells and supernatants collected for VWF to use in experiments. VWF variants containing point mutations at specific sites were constructed via site directed mutagenesis and expressed as above. Myosin from rabbit skeletal muscle was plated on maleic anhydride plates, recombinant VWF or plasma samples added and presence of VWF detected using anti-VWF monoclonal antibodies. Competition assays were performed with antibodies blocking various sites on VWF. FVIII activity was measured using a chromogenic substrate (Chromogenix Coatest). Thrombin generation was performed using a fluorogenic substrate from the Technothrombin TGA kit with either tissue factor +/- myosin or the RCL reagent +/- myosin added to platelet poor plasma from healthy controls.Results: WT VWF and human plasma VWF from healthy controls bound myosin, while plasma lacking VWF failed to demonstrate binding. Binding was multimer dependent, with a dose dependent decrease in binding seen with increasing loss of high molecular weight multimers. When myosin binding to VWF:Ag ratios were compared, a solution of ultra high molecular weight multimers demonstrated a mean ratio of 1.6, a solution of high molecular weight multimers a ratio of 1.4, a solution of medium molecular weight multimers a ratio of 0.4 and binding was undetectable with only low molecular weight multimers. A polyclonal anti-VWF antibody (DAKO) completely blocked VWF binding to myosin as did an antibody directed against the VWF A1 domain (AVW-3). Antibodies directed against other VWF sites including the N and C terminal ends failed to affect VWF binding to myosin. Since collagen IV also binds in this region, collagen IV was added to the assay as competition and also completely blocked VWF binding to myosin. VWF variants p.R1395A and p.R1399A showed undetectable binding, while a variant in the A3 domain (that abrogates type III collagen binding) showed normal binding to myosin (95% of WT). However, additional residues that affect VWF-collagen IV interactions were tested and had minimal effect on myosin binding, including p.1392A and p.1402A. Taken together, these results suggest a binding site for myosin in the VWF A1 domain similar but not identical to that of collagen IV.FVIII activity was detected when a VWF concentrate containing both VWF and FVIII was bound to myosin, with a dose-dependent increase in activity. However, no increase in FVIII activity was seen using recombinant FVIII alone in the absence of additional VWF. Because previous results from J. Griffin and coworkers showed a role for myosin in enhancing thrombin generation, we also looked at thrombin generation in platelet-poor plasma with and without myosin, but no difference was seen in the absence of additional phospholipids. Peak thrombin with myosin was 80 nM vs 62 nM without myosin (p=NS), and the area under the curve was 1022 vs 775 (p=NS). With additional phospholipids, there was a trend towards increased thrombin generation with myosin but the difference was again not statistically significant. Peak thrombin with myosin was 70 nM vs 64 nM without myosin (p=NS), and the area under the curve was 974 vs 867 (p=NS).Discussion: Myosin can also serve as a surface for VWF binding. This may help facilitate delivery of FVIII to sites of injury and improve thrombin generation, although in our hands myosin did not substantially increase thrombin generation in the presence of additional phospholipid. Unlike factor V, FVIII does not intrinsically interact with myosin to accelerate thrombin generation. However, myosin, similar to collagen, can increase local accumulation of VWF-FVIII and indirectly accelerate thrombin generation and clot formation. DisclosuresMontgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

Extracellular Vimentin/VWF (von Willebrand Factor) Interaction Contributes to VWF String Formation and Stroke Pathology.

Background and Purpose- VWF (von Willebrand factor) strings mediate spontaneous platelet adhesion in the vascular lumen, which may lead to microthrombi formation and contribute to stroke pathology. However, the mechanism of VWF string attachment at the endothelial surface is unknown. We tested the novel hypothesis that VWF strings are tethered to the endothelial surface through an interaction between extracellular vimentin and the A2 domain of VWF. We further explored the translational value of blocking this interaction in a model of ischemic stroke. Methods- Human endothelial cells and pressurized cerebral arteries were stimulated with histamine to elicit VWF string formation. Recombinant proteins and antibodies were used to block VWF string formation. Mice underwent transient middle cerebral artery occlusion with reperfusion. Just before recanalization, mice were given either vehicle or A2 protein (recombinant VWF A2 domain) to disrupt the vimentin/VWF interaction. Laser speckle contrast imaging was used to monitor cortical perfusion. Results- Pressurized cerebral arteries produced VWF strings following histamine stimulation, which were reduced in arteries from Vim KO (vimentin knockout) mice. VWF string formation was significantly reduced in endothelial cells incubated with A2 protein or antivimentin antibodies. Lastly, A2 protein treatment significantly improved cortical reperfusion after middle cerebral artery occlusion. Conclusions- We provide the first direct evidence of cerebral VWF strings and demonstrate that extracellular vimentin significantly contributes to VWF string formation via A2 domain binding. Lastly, we show that pharmacologically targeting the vimentin/VWF interaction through the A2 domain can promote improved reperfusion after ischemic stroke. Together, these studies demonstrate the critical role of VWF strings in stroke pathology and offer new therapeutic targets for treatment of ischemic stroke.

Delimiting the autoinhibitory module of von Willebrand factor

Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP)Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Destabilization of the von Willebrand factor A2 domain under oxidizing conditions investigated by molecular dynamics simulations

The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib α (GpIbα). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIbα.

Assessment of Novel Antioxidant Therapy in Atherosclerosis by Contrast Ultrasound Molecular Imaging

Ultrasound molecular imaging was used to evaluate the therapeutic effects of antioxidant therapy with EUK-207, which has superoxide dismutase and catalase activities, on suppressing high-risk atherosclerotic features. Mice with age-dependent atherosclerosis produced by deletion of the low-density lipoprotein receptor and Apobec-1 were studied at 20 and 40weeks of age. EUK-207 or vehicle was administered for the preceding 8weeks. Therapy for 28weeks was also studied for 40-week-old mice. Ultrasound molecular imaging of the thoracic aorta was performed with contrast agents targeted to endothelial P-selectin, von Willebrand factor A1-domain, and platelet glycoprotein Ibα or control agent. Aortic plaque area and macrophage content were assessed by histology. In 20-week-old double-knockout mice, EUK-207 compared with sham therapy produced only nonsignificant trends for reduction in molecular imaging signal for endothelial P-selectin, von Willebrand factor A1-domain, and platelet adhesion. At 40weeks, EUK-207 given for 8 or 28weeks significantly (P<.05) reduced signal for all three endothelial-associated events essentially to background levels, with the exception of glycoprotein Ibα signal after 8weeks (P=.06). On aortic histology, EUK-207 therapy for 8weeks did not affect plaque area or macrophage content at either age. However, EUK-207 for 28weeks almost completely suppressed plaque development (350±258 vs 4±6×103μm2, P=.014) and macrophage content (136±103 vs 3±2×103μm2, P=.002) compared with control mice at 40weeks. Molecular imaging can be used to assess vascular responses to antioxidants and has demonstrated that certain antioxidants reduce vascular endothelial activation and platelet adhesion, but reductions in plaque size and macrophage content occurs only with long-duration therapy that is started early.

Myocardial Infarction Produces Sustained Proinflammatory Endothelial Activation in Remote Arteries

BackgroundIn the months after acute myocardial infarction (MI), risk for acute atherothrombotic events in nonculprit arteries increases several fold. ObjectivesThis study investigated whether sustained proinflammatory and prothrombotic endothelial alterations occur in remote vessels after MI. MethodsWild-type mice, atherosclerotic mice with double knockout (DKO) of the low-density lipoprotein receptor and Apobec-1, and DKO mice treated with the Nox-inhibitor apocynin were studied at baseline and at 3 and 21 days after closed-chest MI. Ultrasound molecular imaging of P-selectin, vascular cell adhesion molecule (VCAM)-1, von Willebrand factor (VWF) A1-domain, and platelet GPIbα was performed. Intravital microscopy was used to characterize post-MI leukocyte and platelet recruitment in the remote microcirculation after MI. ResultsAortic molecular imaging for P-selectin, VCAM-1, VWF-A1, and platelets was increased several-fold (p < 0.01) 3 days post-MI for both wild-type and DKO mice. At 21 days, these changes resolved in wild-type mice but persisted in DKO mice. Signal for platelet adhesion was abolished 1 h after administration of ADAMTS13, which regulates VWF multimerization. In DKO and wild-type mice, apocynin significantly attenuated the post-MI increase for molecular targets, and platelet depletion significantly reduced P-selectin and VCAM-1 signal. On intravital microscopy, MI resulted in remote vessel leukocyte adhesion and platelet string or net complexes. On histology, high-risk inflammatory features in aortic plaque increased in DKO mice 21 days post-MI, which were completely prevented by apocynin. ConclusionsAcute MI stimulates a spectrum of changes in remote vessels, including up-regulation of endothelial inflammatory adhesion molecules and platelet-endothelial adhesion from endothelial-associated VWF multimers. These remote arterial alterations persist longer in the presence of hyperlipidemia, are associated with accelerated plaque growth and inflammation, and are attenuated by Nox inhibition.

Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF

Collagen-based scaffolds may require chemical crosslinking to achieve mechanical properties suitable for tissue engineering. Carbodiimide treatment, often used for this purpose, consumes amino acid side chains required for receptor recognition, thus reducing cell–collagen interaction. Here, we restore recognition and function of both von Willebrand Factor (VWF) and Discoidin Domain Receptor 2 (DDR2) to crosslinked collagen films by derivatisation with a specific triple-helical peptide (THP), an approach previously applied to integrin-mediated cellular adhesion. The THP contained the collagen III-derived active sequence, GPRGQOGVNleGFO, conjugated to a photoreactive moiety, diazirine, allowing UV-dependent covalent coupling to collagen films. Crosslinking of collagen films attenuated the binding of recombinant VWF A3 domain and of DDR2 (as the GST and Fc fusions, respectively), and coupling of the specific THP restored their attachment. These derivatised films supported activation of DDR2 expressed in either COS-7 or HEK293 cells, reflected by phosphorylation of tyrosine 740, and VWF-mediated platelet deposition from flowing blood was restored. Further, such films were able to increase low-density lipoprotein uptake in vascular endothelial cells, a marker for endothelial phenotype. Thus, covalent linkage of specific THPs to crosslinked collagen films i) restores their cognate protein binding, ii) triggers the corresponding cellular responses, and iii) demonstrates the broad applicability of the approach to a range of receptors for applications in regenerative medicine.

Cellular stress induces erythrocyte assembly on intravascular von Willebrand factor strings and promotes microangiopathy

Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.

Von Willebrand Factor for Aortic Valve Intervention: From Bench to Real-Time Bedside Assessment.

VWF (von Willebrand factor ) is a circulating multimeric blood glycoprotein. VWF plays a major role in primary hemostasis by promoting the adhesion of platelets to subendothelial collagen at sites of vascular damage and thereby promoting platelet aggregation. VWF is synthesized in endothelial cells and megakaryocytes. The VWF units dimerize and are transported into the Golgi apparatus, where disulfide bonds are formed leading to formation of VWF multimers. This large subunit combination is required to support its hemostatic function. VWF has the unique features to be circulating in an inactive coiled form, hiding binding domains for platelet receptors and subendothelial collagen. At the site of vascular injury, VWF binds to the exposed collagen and unfolds. Once VWF is unfolded, the VWF A1 domain is exposed allowing the binding of platelets via GP Ib (glycoprotein IB) receptor. After platelet activation, GP IIb/IIIa (glycoprotein IIb/IIIa) receptor becomes able to bind VWF C1 domain. The VWF conformation and activity is intimately related to shear conditions and blood flow. At high shear rate (beyond 10–15 pN), it becomes unfolded exposing binding sites but also the cleavage site in VWF A2 domain for ADAMTS13 (adisintegrin-like and metalloprotease thrombospondin) protease that conducts to its proteolysis. Overall, these environmental changes generate the modification of the conformation of VWF …

Describing the molecular mechanism by which the A2 protein improves survival in mice with endotoxemia.

Von Willebrand factor (VWF) is an essential protein that mediates the platelet adhesion to the exposed subendothelium surface and significantly contributes to arterial thrombosis and stroke. Secreted VWF from the stimulated endothelium as long strings remain anchored to the surface of the endothelial cells and interact with circulating platelets, leading to vessel occlusion. We have shown that the isolated A2 domain of VWF (A2 protein) contains bind activity for the A1 domain, vimentin, and fibrin(ogen). In fact, as a result of those interactions, the A2 protein impairs platelet adhesion to VWF and fibrin(ogen), formation of VWF strings on the endothelium, and fibrin polymerization. The A2 protein was effective in preventing fibrin‐rich microvascular thrombosis and improving survival (100%) of mice with endotoxemia (LPS)‐induced disseminated intravascular coagulation (DIC). Thus, a goal was to identify the binding sites of A1 domain, vimentin, and fibrin in the A2 protein and dissect the mode of action by which the A2 protein recuperates the sick animals. To determine the amino acid residues that are important in the recognition for vimentin, A1 and fibrin, I used the crystal structure of the A2 domain as a guide for point mutations. Two mutations, E1523A and E1567A were introduced in the A2 protein. The purified mutants were tested for their binding capacity for the ligands using ELISA. Moreover, the A2 mutants were tested in mice with endotoxemia (LPS). We are now demonstrating that E1523A mutation had a reduced binding affinity for Vimentin and A1 domain. Strikingly, E1567A and E1523A mutations significantly abolished the binding to fibrin(ogen). Importantly, both mutations failed to replicate the beneficial effect of the wild type A2 protein in improving survival of mice with LPS‐induced DIC. Together, these findings suggest that the A2 protein impairs microvascular thrombosis in mice with endotoxemia mainly via its interaction with fibrin.Support or Funding InformationThis research was supported by funding provided by the following NIH Diversity Supplement: R01GM112806‐0351.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

"Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how and the urea m-value interconvert through the slope of cm versus T, . This relationship permits the calculation of at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

A common mechanism by which type 2A von Willebrand disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand factor A2 domain FRET construct.

Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi

BackgroundThe bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3β1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made.ResultsOur results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions.ConclusionsPresently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.