Von Willebrand Factor A1 Research Articles

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.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.

RNA-Seq reveals a central role for lectin, C1q and von Willebrand factor A domains in the defensive glue of a terrestrial slug

The tough, hydrogel glue produced by the slug Arion subfuscus achieves impressive performance through metal-based, protein cross-links. The primary sequence of these proteins was determined through transcriptome sequencing and proteome analysis by tandem mass spectrometry. The main proteins that correlate with adhesive function are a group of 11 small, highly abundant lectin-like proteins. These proteins matched the ligand-binding C-lectin, C1q or H-lectin domains. The variety of different lectin-like proteins and their potential for oligomerization suggests that they function as versatile and potent cross-linkers. In addition, the glue contains five matrilin-like proteins that are rich in von Willebrand factor A (VWA) and EGF domains. Both C-lectins and VWA domains are known to bind to ligands using divalent cations. These findings are consistent with the double network mechanism proposed for slug glue, with divalent ions serving as sacrificial bonds to dissipate energy.

A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Lighting up kinase action in platelets

Platelet functions are highly regulated by signaling networks involving at least 300 protein kinases [2]. Platelets express members of the mitogen-activated protein kinase (MAPK) family including p42 ERK2 and, at lower concentrations, p44 ERK1. Both ERK isoforms are activated by agonists like von Willebrand factor, collagen, thrombin, ADP and thromboxane A2 [3-7]. ERK activation depends on MAPK kinase (MKK-1 or MEK) and involves Gq, Gi, phospholipase C and Src family kinase signaling [5, 8]. Active ERK has been shown to stimulate thromboxane A2 production [5] and to contribute to platelet aggregation [4, 9]. Downstream substrates of ERK have not been identified in platelets so far. This article is protected by copyright. All rights reserved.

A Novel Single-Domain Antibody Against von Willebrand Factor A1 Domain Resolves Leukocyte Recruitment and Vascular Leakage During Inflammation-Brief Report.

von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo. Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage. KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF-platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response.

Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.

Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin. We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain. Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability.

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von-Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long-term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (∼300nM), which is significantly weaker than that between full-length PA and CMG2 (170-300pM). Unlike full-length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or 19 F-NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ∼10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.

A Novel Monoclonal Antibody Against A2 Domain of Von Willebrand Factor Reduces Its Proteolysis By ADAMTS13

Introduction:ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. A minimal region as a functional substrate for ADAMTS13 consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and deletion of the E1660-R1668 region (the C-terminal α helix of VWF A2 domain) led to the loss of cleavage by ADAMTS-13. This suggested that the C-terminal α helix of VWF A2 domain contributes to ADAMTS13 binding to substrate. We will explore whether murine monoclonal antibody (mAb) against the C-terminal α helix of human VWF A2 domain affects the susceptibility of VWF to proteolysis by ADAMTS13 in vitro.Methods:Balb/c mice were immunized with R1659-R1668 of the VWF polypeptide. Monoclonal antibodies (mAbs) were developed by standard hybridoma technology and identified with ELISA. The epitope of mAbs were mapped using recombinant VWF A2 and a series of its truncated mutants with western blot analysis. The effect of mAbs to VWF on its proteolysis by ADAMTS13 was investigated under static/denaturing condition by 1.3% agarose gel electrophoresis and immunologic analysis. The effect of mAbs to GST-VWF73-His on its proteolysis by recombinant ADAMTS13 was measured by 15% SDS-PAGE under reducing condition and western blot analysis.Results: One mAb, designated SZ-179, was found to have high affinity (50 ng/ml) with both a synthetic peptide encompassing residues 1659-1668 and purified pVWF. SZ-179 has isotype of IgG1. It was shown that SZ-179 reacted specifically with recombinant VWF A2 and GST-VWF73-His, respectively, but not with recombinant VWF A1 nor with recombinant VWF A3. Epitope mapping experiments revealed that SZ-179 bound to the distal portion of the VWF A2 domain (E1660-L1666). SZ-179 inhibited proteolytic cleavage of GST-VWF73-His by recombinant ADAMTS13 in a concentration-dependent manner, with a maximal inhibition at concentrations of 50 ug/ml. SZ-179 also reduced degradation of high molecular-weight (HMW)-VWF-multimers under static/denaturing condition dose-dependently, with a maximal inhibition at concentrations of 1 ug/ml.Conclusion:SZ-179, a novel mAb to the A2 Domain of VWF, inhibits the interactions between VWF and ADAMTS13, and prevents excessive degradation of HMW-VWF multimers. This mAb might be useful for the development of therapeutic options to treat bleeding episodes among hemorrhagic diseases. DisclosuresNo relevant conflicts of interest to declare.

A Short N-Terminal Fragment in Front of the A1 Domain of Von Willebrand Factor Prevents the A1 from Binding the Platelet By Masking a Region Clustered By Type 2B VWD Mutations

Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb.We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region.Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of Platforms to Phenotype Variants of Uncertain Significance in VWF

Platelet sequestration at sites of vascular injuries and factor VIII (FVIII) stabilization critically relies on von Willebrand factor (VWF). Specific domains within VWF coordinate these functions and may harbor mutations that result in von Willebrand disease (VWD). To survey all possible mutations that may lead to type 2 VWD, we developed prokaryotic and eukaryotic systems to display VWF fragments that are screened for a specific function and identified by DNA sequencing. We constructed an M13 filamentous phage display library consisting of ~2.8x106 independent clones that express random VWF fragments. Following a single round of selection for platelet binding, bound phage were eluted and analyzed by next generation DNA sequencing, revealing an optimal fragment spanning the A1 domain and a second region of weaker enrichment over the D4 domain. All A1 fragments encompassed C1272-C1458, indicating that intramolecular disulfide bridging of these cysteines optimizes platelet binding. Competitive binding assays between phage displaying a fragment of the D4 domain vs. a control phage resulted in enrichment of the former, suggesting a potential, novel role for the VWF D4 domain. Analogous to our previously reported method for analyzing VWF proteolysis (Kretz CA et al. Proc Natl Acad Sci USA. 2015), we mutagenized and displayed the VWF A1 domain to determine the spectrum of mutations that may alter the interaction between VWF and its platelet receptor, GPIbα. The mutant A1 library comprised ~5x106 independent clones, sufficient to represent all 3,933 single substitutions. Screening this library against platelets in the presence of excess control phage resulted in an enrichment of phage displaying an A1 fragment. DNA sequencing identified mutations located outside the VWF A1/platelet GPIbα interface, indicating allosteric control of this interaction. Screening phage displayed VWF fragments for FVIII binding, however, failed to identify critical residues; thus, we adapted our approach for eukaryotes. When displayed on mammalian cells, the VWF D’D3 domains retained their capacity to bind FVIII. Introduction of the VWD subtype 2N mutation, R816W, into the displayed VWF D’D3 domains abolished FVIII binding, demonstrating feasibility for mutational analyses. Together, these results demonstrate the utility of phage and mammalian display for structure-function analyses and that these platforms provide an efficient method for phenotyping variants of uncertain significance. DisclosuresNo relevant conflicts of interest to declare.

Phylogenetic Analysis Identifies a Subset of ADAMTS13 Domains That Are Highly Conserved and Essential for Allosteric Regulation

Introduction: Human ADAMTS13 has metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB domains (distal domains).ADAMTS13 regulates von Willebrand factor (VWF) by cleaving a Tyr-Met bond in the VWF A2 domain that is exposed under shear force. Acquired or inherited ADAMTS13 deficiency impairs this regulatory mechanism and cause thrombotic thrombocytopenic purpura (TTP), a life threatening disorder. We recently showed that ADAMTS13 is autoinhibited by its distal T8-CUB domains and this inhibition is relieved by binding to VWF. Thus, VWF promotes its own destruction by allosterically activating ADAMTS13. We investigated the evolution and conservation of this remarkable regulatory mechanism.Methods:ADAMTS13 cDNA sequences for >300 species were assembled from genome assemblies and SRA transcriptomic and genomic data. Recombinant ADAMTS13 variants were expressed in HEK293 cells and purified chromatographically. Plasma was obtained from dog, armadillo, rabbit, primates (human, baboon, marmoset, macaque), rodents (rat, mouse), ungulates (cattle, sheep, pig, horse), birds (chicken, pigeon), and Xenopus laevis. Plasma ADAMTS13 activity of at least three members of each species was assayed with human-FRETS71, a fluorogenic ADAMTS13 substrate based on human VWF. Similar fluorogenic substrates were prepared with armadillo, rat, pigeon and cattle VWF sequences. Plasma ADAMTS13 also was assayed with allosteric activators of human ADAMTS13: monoclonal antibodies (MAbs) against distal T and CUB domains, low pH (pH 6), and recombinant human VWF D4 domain. ADAMTS13-VWF interactions were assessed with biolayer interferometry (BLI) and binding data fitted to 1:1 binding model.Results: The ancestral ADAMTS13 had a long propeptide that is characteristic of most metalloproteases but notably missing from human ADAMTS13, and 8 distal T domains compared to 7 distal T domains in human ADAMTS13. The ancestral structure today is found in most reptiles, amphibians, and marsupials. The propeptide, T3, T4, T6 and T6a have been deleted independently several times in mammals, birds, amphibians and fish. Most animal plasmas cleaved human-FRETS71 less rapidly than human plasma, although armadillo, rabbit, and dog were 2.4-, 3.0-, 4.0-fold more active, respectively. Human and cattle VWF have Tyr-Met cleavage sites, but armadillo VWF has a Tyr-Leu bond. Armadillo plasma cleaved armadillo-FRETS71 and human-FRETS71 at a similar rate; other species cleaved armadillo-FRETS71 15% to 88% as rapidly as human-FRETS71. Surprisingly, cattle-FRETS71 was cleaved ~2- to 15-fold faster than human-FRETS71 by most plasmas. Except for C57Bl/6 mice (which have ADAMTS13 truncated after T6), armadillo, and dog, all plasmas screened were activated by MAbs or VWF D4. Frog and pigeon ADAMTS13 were activated ~6-fold by D4, whereas human ADAMTS13 was activated only 2-fold. Absence of CUB domains in armadillo ADAMTS13 abolished activation by D4. In contrast, absence of all but 3 distal T domains in pigeon ADAMTS13 did not impair activation by VWF D4. Recombinant human ADAMTS13 constructed with deletions like those in armadillo and pigeon ADAMTS13 had similar properties: delT3-6 was activated by VWF D4 but M-T8 was not. All enzymes activated by VWF D4 showed enhanced cleavage of multimeric VWF under fluid shear stress compared to enzymes that are not activated by VWF D4 (Figure 1 and Table 1). When normalized for activity toward FRETS71, pigeon ADAMTS13 was ~2-fold more specific for VWF multimers than is human ADAMTS13. In agreement with the cleavage data, recombinant human and pigeon ADAMTS13, and MT-8 bound VWF D4-CK with Kd values in the 350-840 nM range, whereas MDTCS, MT-7 and delT2-7 did not bind.Conclusion: Allosteric regulation of ADAMTS13 by VWF is broadly conserved, with some apparent exceptions. The distal T3-T6 domains are dispensable for allosteric regulation, whereas T7, T8 and CUB domains are required. Furthermore, ADAMTS13 from different species exhibits striking variation in substrate specificity. For most species, altering the sequence of the VWF substrate can markedly increase the rate of cleavage, indicating that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties are likely to reflect evolutionary pressure to balance the risk of VWF-dependent bleeding and thrombosis. [Display omitted] [Display omitted] DisclosuresSadler:Ablynx: Consultancy; 23andMe: Consultancy; BioMarin: Consultancy.

A Quantitative Assay of In Vivo Cleavage of Von Willebrand Factor By ADAMTS13 Reveals Excessive Cleavage in Type 2B Von Willebrand Disease and Diminished Cleavage in Thrombotic Thrombocytopenic Purpura

Von Willebrand Factor (VWF) is a large glycoprotein that plays a crucial role in hemostasis by enabling platelets to adhere to sites of vascular injury, particularly under conditions of high shear stress. The multimeric structure of VWF is central to its activity; VWF is most active in its ultra-large multimer form (ULVWF). The size of plasma VWF is regulated by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Tyr1605-Met1606 peptide bond within the A2 domain. Abnormal VWF cleavage has been implicated in a wide range of thrombotic and bleeding disorders, including thrombotic thrombocytopenic purpura (TTP) and Type 2 von Willebrand disease (VWD). These two diseases represent extreme phenotypes of VWF proteolysis-the former with too little cleavage and the latter with too much cleavage. ADAMTS13 proteolysis of VWF is currently assessed by either multimer gel analysis or ADAMTS13 activity assays. High-resolution agarose gels give an estimate of the extent to which a particular band represents cleaved vs native VWF. This method, however, is not quantitative and cannot distinguish whether a particular cleavage product is produced by ADAMTS13 or by another protease. Alternatively, ADAMTS13 activity assays are indirect measures of VWF cleavage, used especially to diagnose TTP. These assays measure the extent to which a subject's ADAMTS13 can cleave an exogenous substrate, usually a small peptide, and do not determine the extent of endogenous VWF cleavage. Therefore, we developed a mass spectrometry-based method to directly quantify cleavage of the Tyr1605-Met1606 peptide bond in plasma VWF. Here, we report quantification of VWF cleavage in plasma from normal donors, and TTP and VWD patients.Methods: VWF was isolated from plasma by immunoprecipitation using polyclonal VWF antibody and then digested with trypsin. Tryptic peptides, including the ADAMTS13-cleaved peptide and a control peptide, were quantified by nano ultra-performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using heavy isotope-labelled peptides as internal standards. The extent of VWF cleavage by ADAMTS13 was determined as the concentration ratio of ADAMTS13-cleaved peptide to a control peptide representative of total VWF, and expressed as percent cleavage.Results: Given that the plasma concentration of VWF is relatively low, we first optimized conditions for sample preparation and analysis. Using NanoLC-MS/MS with isotopic dilution, we quantified ADAMTS13 cleavage of VWF in plasma samples (10-100 µl) containing 100-500 ng of VWF. We tested the reproducibility of the measurement using pooled normal plasma. The quantification was highly reproducible, with a wide linear range. VWF cleavage in pooled normal plasma was 4.9% ± 0.3% (mean ± SD, n=6), indicating that one VWF monomer in 20 is cleaved, on average. Cleavage was much higher in plasma from type 2B VWD patients (n=5), ranging from 15-30% depending on mutations. In contrast, we observed only 2.1-3.2% (n=6) cleavage in plasma VWF from TTP patients.Summary: This is the first report of quantification of ADAMTS13-mediated VWF cleavage in plasma. In normal plasma, approximately 5% of VWF A2 domains are cleaved, whereas cleavage was reduced to approximately 60% of normal on average in 6 TTP patients, and increased up to 6 times normal in patients with Type 2B VWD. In TTP, this level of proteolysis would be expected to result in accumulation of ULVWF multimers, as only between 1 in 50 to 1 in 30 monomers were cleaved. In type 2B VWD, by contrast, proteolysis may play a much larger role in the lack of larger multimers than previously appreciated, as up to 1 in 3 monomers were cleaved. This method should also prove useful in conditions such as severe malaria, in which it has been suggested that VWF cleavage is impaired, but this is not necessarily accompanied by low ADAMTS13 activity as measured with a small peptide substrate. DisclosuresNo relevant conflicts of interest to declare.

Potential Mechanisms for Enhanced Activity of Von Willebrand Factor in Patients with Sickle Cell Disease

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusion and hemolysis. Even at disease baseline, von Willebrand factor (VWF) quantity and activity is markedly increased and correlates with the extent of hemolysis [Chen et al. Blood, 2011, 117:3680].VWF is an adhesive plasma protein capable of binding platelets, and secondarily, erythrocytes, and leukocytes. Its adhesive activity is regulated at several levels: being decreased by proteolysis by the metalloprotease ADAMTS13 [Dong et al. Blood, 2002, 100:4033] or by inhibition of self-association into more adhesive forms by high density lipoprotein (HDL) [Chung et al. Blood, 2016, 127:637], or enhanced by oxidative modification [Chen et al. Blood, 2010, 115:706; Fu et al. Blood, 2011, 118:5283]. Here, we explored several potential mechanisms accounting for the increased total active VWF in SCD patients: endothelial activation, enhanced VWF self-association, and defective cleavage of endogenous VWF because of ADAMTS13 dysfunction and/or VWF resistance to cleavage due to oxidation.Methods: We analyzed plasma collected from 12 adult SCD patients at disease baseline; for 3 of these we also have samples collected during vaso-occlusive crisis. We also analyzed multiple crisis samples from one additional patient with no baseline sample. To determine if SCD plasma could activate endothelial cells, we incubated plasma with human umbilical vein endothelial cells (HUVECs) cultured in parallel-plate flow chambers for 20 min at 37˚C then washed the plasma away before perfusing fixed platelets through the chamber. The number and length of platelet-decorated VWF strings were then quantified as a measure of endothelial activation. The plasma levels of VWF, apolipoprotein A-I (ApoA-I, the major apolipoprotein in HDL), myeloperoxidase (MPO), and ADAMTS13 were measured by sandwich ELISA. The activity of ADAMTS13 was evaluated by 1) cleavage of a small VWF A2 peptide substrate; 2) cleavage of multimeric VWF in patient plasma in 1.5 M urea and examining the VWF multimer pattern over time; 3) cleavage of preformed VWF-platelet strings on activated HUVECs; and 4) examining the extent of VWF cleavage in vivo using mass spectrometry (MS). The oxidation of specific methionine residues in VWF and ADAMTS13 from patient plasma was measured by MS as described previously [Chen et al. Blood, 2010, 115:706; Wang et al. JBC, 2015, 290:1422].Results: 1) HUVECs incubated with SCD plasma were activated and produced 2 to 25 fold more VWF strings than cells treated with pooled normal plasma (PNP). Activation was inhibited if the plasma was pre-incubated with hemopexin (0.5 mg/ml), implicating heme as an activator. However, purified heme alone even at high concentration did not activate HUVECs to secrete VWF strings, indicating that heme is required but not sufficient. 2) The ratio of VWF to ApoA-I was markedly elevated in SCD patients, especially during crisis (2.3 ± 0.7 fold of PNP), indicating predisposition to VWF self-association. 3) ADAMTS13 from SCD plasma was defective in cleaving VWF strings (11% to 57% of PNP) and multimeric VWF, but not the A2 peptide substrate. 4) Almost all of the SCD plasma had elevated MPO (3.8 ± 2.5 and 12.8 ± 7.6 fold of PNP in baseline and crisis samples, respectively). 5) Our previous studies showed that HOCl oxidation of VWF Met1606 at the ADAMTS13 cleavage site rendered the VWF noncleavable, and oxidation at other sites increased its platelet-binding activity. We also found that HOCl can inactivate ADAMTS13 by oxidizing Met249 within the metalloprotease domain. We therefore evaluated the extent of VWF Met1606 and ADAMTS13 Met249 oxidation, and VWF cleavage by ADAMTS13 in vivo in patient plasma using tandem MS. We found that ADAMTS13 in SCD plasma had elevated oxidation at Met249 (1.6 ± 0.2 fold of normal controls); VWF cleavage by ADAMTS13 in vivo was decreased by as much as 40% in several of these patients, and the extent of cleavage correlated negatively with the extent of VWF Met1606 oxidation.Summary: Multiple mechanisms contribute to enhanced VWF activity in SCD patients. These include increased secretion by activated endothelial cells, enhanced VWF self-association, and defective cleavage of VWF by endogenous ADAMTS13, partially due to oxidative modification of both VWF and ADAMTS13. DisclosuresNo relevant conflicts of interest to declare.

The Von Willebrand Factor A1–Collagen III Interaction Is Independent of Conformation and Type 2 Von Willebrand Disease Phenotype

The blood von Willebrand factor (VWF) mediates platelet adhesion to injured vessels by sequestering platelets from blood flow and depositing them to collagen and other exposed subendothelial matrix proteins. This process of capturing platelets to facilitate formation of platelet plugs occurs through transient interactions with platelet glycoprotein Ibα via the VWF A1 domain which also binds collagen. Using a conformationally diverse collection of natively folded and mutation-induced misfolded von Willebrand disease (VWD) variants, we test a recently proposed affinity up-regulation hypothesis which states that collagen binding changes the conformation of the A1 domain to a high-affinity GPIbα binding competent state. With surface plasmon resonance (SPR), we present this diversified collection to collagen and quantify the kinetics of association and dissociation to ascertain the conformational selectivity of collagen. With analytical rheology, we quantify real-time platelet pause times and translocation velocities across a Cu2+ HisTag-chelated and collagen-bound A1 single domain and A1A2A3 tridomain fragment of VWF under shear stress in an ex vivo shear flow microfluidic chamber. In contrast to expected hypothetical outcomes, collagen has limited conformational selectivity for binding A1. A1-collagen binding is independent of gain- or loss-of-function phenotype and under shear stress, platelet translocation pause times on collagen-bound A1A2A3 are either normal or shorter depending on whether A1 is concertedly bound with the A3 domain to collagen. With respect to A1, collagen has an inhibitory role that provides an explanation for the lack of thrombosis in patients with gain-of-function VWD.

Biophysical approaches promote advances in the understanding of von Willebrand factor processing and function.

The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion. High-resolution stochastic optical reconstruction microscopy, atomic force microscopy (AFM), microscale thermophoresis and fluorescence correlation spectroscopy allowed identification of protein disulfide isomerase isoform A1 as the VWF dimerizing enzyme and, together with molecular dynamics simulations, postulation of the dimerization mechanism. Advanced mass spectrometry led to detailed identification of the glycan structures carried by VWF. Microfluidics was used to illustrate the interplay of force and VWF function. Results from optical tweezers measurements explained mechanisms of the force-dependent functions of VWF's domains A1 and A2 and, together with thermodynamic approaches, increased our understanding of mutation-induced dysfunctions of platelet-binding. AFM-based force measurements and AFM imaging enabled exploration of intermonomer interactions and their dependence on pH and divalent cations. These advances would not have been possible by the use of biochemical methods alone and show the benefit of interdisciplinary research approaches.

Type 2B von Willebrand Disease: A Matter of Plasma Plus Platelet Abnormality.

Type 2B von Willebrand disease (VWD2B) is a rare, autosomal-dominant inherited bleeding disorder, characterized by an enhanced ristocetin-induced platelet aggregation in platelet-rich plasma and often with variable degree of thrombocytopenia and loss of high-molecular-weight multimers von Willebrand factor (VWF). All these phenomena are caused by a mutant VWF, normally synthesized and assembled by endothelial cells, but with heightened affinity binding to the platelet receptor glycoprotein Ib-α (GpIb-α). When this abnormal VWF is released into the circulation and under specific clinical circumstances, in vivo platelet clumping is observed. Mutations, invariably clustered in exon 28 of the VWF gene encoding for the VWF A1 domain involved in VWF binding to GpIb-α, are responsible for VWD2B phenotype. Clinical and laboratory phenotype appears strongly related to the type of VWF-causative mutations. However, recent evidences suggest that a true platelet defect is also present in this type, with several morphological and functional abnormalities being detected in a subset of VWD2B patients.

N-linked glycan stabilization of the VWF A2 domain

AbstractShear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca2+ binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca2+ binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.

Untangling a Structurally Resolved Protein Folding Intermediate

A central challenge in protein biophysics is to decipher the structural features that give rise to the energetics of protein folding pathways and determine structural details of intermediates. A unique advantage of single-molecule force spectroscopy over classical thermodynamic methods of monitoring protein folding and unfolding transitions is that force spectroscopy effectively reduces the ensemble of possible conformational states to those whose unfolding trajectories share a common reaction coordinate experimentally controlled by the vector of applied force.