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

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

The von Willebrand Factor A-1 domain binding aptamer BT200 elevates plasma levels of VWF and Factor VIII: a first-in-human trial

Von Willebrand Factor (VWF) and Factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary haemostasis and clotting respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomised, placebo-controlled, double-blind trial tested the hypothesis that BT200 is well tolerated and has favourable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: Single ascending dose of BT200 (0.18-48mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterised, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin induced aggregation, platelet function under high shear rates, and thrombin generation. Mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dosedependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagenadenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (p

Staphylococcus aureus iron-regulated surface determinant B (IsdB) protein interacts with von Willebrand factor and promotes adherence to endothelial cells

Staphylococcus aureus is the cause of a spectrum of diseases in humans and animals. The molecular basis of this pathogenicity lies in the expression of a variety of virulence factors, including proteins that mediate adherence to the host plasma and extracellular matrix proteins. In this study, we discovered that the iron-regulated surface determinant B (IsdB) protein, besides being involved in iron transport and vitronectin binding, interacts with von Willebrand Factor (vWF). IsdB-expressing bacteria bound to both soluble and immobilized vWF. The binding of recombinant IsdB to vWF was blocked by heparin and reduced at high ionic strength. Furthermore, treatment with ristocetin, an allosteric agent that promotes the exposure of the A1 domain of vWF, potentiates the binding of IsdB to vWF. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound recombinant A1 domain with KD values in the micromolar range. The binding of IsdB and adhesion of S. aureus expressing IsdB to monolayers of activated endothelial cells was significantly inhibited by a monoclonal antibody against the A1 domain and by IsdB reactive IgG from patients with staphylococcal endocarditis. This suggests the importance of IsdB in adherence of S. aureus to the endothelium colonization and as potential therapeutic target.

Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library. Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin. Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267). When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12. Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Staphylococcus Aureus Iron-Regulated Surface Determinant B (IsdB) Protein Interacts with von Willebrand Factor and Promotes Adherence to Endothelial Cells.

Staphylococcus aureus is the cause of a spectrum of diseases in humans and animals. The molecular basis of this pathogenicity lies in the expression of a variety of virulence factors, including proteins that mediate adherence to the host plasma and extracellular matrix proteins. In this study, we discovered that the iron-regulated surface determinant B (IsdB) protein, besides being involved in iron transport and vitronectin binding, interacts with von Willebrand Factor (vWF). IsdB-expressing bacteria bound to both soluble and immobilized vWF. The binding of recombinant IsdB to vWF was blocked by heparin and reduced at high ionic strength. Furthermore, treatment with ristocetin, an allosteric agent that promotes the exposure of the A1 domain of vWF, potentiates the binding of IsdB to vWF. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound recombinant A1 domain with KD values in the micromolar range. The binding of IsdB and adhesion of S. aureus expressing IsdB to monolayers of activated endothelial cells was significantly inhibited by a monoclonal antibody against the A1 domain and by IsdB reactive IgG from patients with staphylococcal endocarditis. This suggests the importance of IsdB in adherence of S. aureus to the endothelium colonization and as potential therapeutic target.

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR (or CD320), encoded by the CD320 gene(1). Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells such as cancer cells(2–4), this uptake route is a potential target for cancer therapy(5). We developed and characterized four camelid nanobodies that bind TC or the interface of the TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of all four nanobodies in complex with TC:TCblR, which enabled us to map their binding sites. When conjugated to a toxin, three of these nanobodies are capable of inhibiting the growth of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TCNB4) using fluorescent light microscopy. The co-crystal structures of TC:TCblR with another nanobody (TCNB34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR. Our findings rationalize the structural basis for a decrease in affinity of TC-B12 binding caused by the TCblR-Glu88 deletion mutant.

The GPIbα intracellular tail – role in transducing VWF- and collagen/GPVI-mediated signaling

The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing both VWF-GPIbGGand collagen-GPVI signaling events in platelets.

Type 2A and 2M von Willebrand Disease: Differences in Phenotypic Parameters According to the Affected Domain by Disease-Causing Variants and Assessment of Pathophysiological Mechanisms

Type 2A and 2M von Willebrand disease (VWD) broadly show similar phenotypic parameters, but involve different pathophysiological mechanisms. This report presents the clinical and laboratory profiles of type 2A and type 2M patients genotypically diagnosed at one large center. Higher bleeding score values and a higher incidence of major bleeding episodes were observed in type 2A compared with type 2M, potentially reflective of the absence of large and intermediate von Willebrand factor (VWF) multimers in 2A. In type 2A, most of disease-causing variants (DCVs) appeared to be responsible for increased VWF clearance and DCV clustered in the VWF-A1 domain resulted in more severe clinical profiles. In type 2M, DCV in the VWF-A1 domain showed different laboratory patterns, related to either reduced synthesis or shortened VWF survival, and DCV in the VWF-A2 domain showed patterns related mainly to shortened survival. VWF-type 1 collagen binding/Ag (C1B/Ag) showed different patterns according to DCV location: in type 2A VWD, C1B/Ag was much lower when DCVs were located in the VWF-A2 domain. In type 2M with DCV in the VWF-A1domain, C1B/Ag was normal, but with DCV in the VWF-A2 domain, C1B/Ag was low. The higher frequency of major bleeding in VWD 2M patients with DCV in the VWF-A2 domain than that with DCV in the VWF-A1 domain could be a summative effect of abnormal C1B/Ag, on top of the reduced VWF-GPIb binding. In silico modeling suggests that DCV impairing the VWF-A2 domain somehow modulates collagen binding to the VWF-A3 domain. Concomitant normal FVIII:C/Ag and VWFpp/Ag, mainly in type 2M VWD, suggest that other nonidentified pathophysiological mechanisms, neither related to synthesis/retention nor survival of VWF, would be responsible for the presenting phenotype.