A1 Domain Research Articles

Cryo-EM structures of human coagulation factors V and Va

Coagulation factor V (fV) is the precursor of fVa, which, together with fXa, Ca2+, and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. We solved the cryogenic electron microscopy (cryo-EM) structures of human fV and fVa at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly, but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding fXa, and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain that is responsible for prothrombin binding. Ordering of this region and full exposure of the fXa epitope emerge as necessary steps in the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of fV and fVa and pioneer the analysis of coagulation factors by cryo-EM.

Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor.

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details of how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ibα (GpIbα), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIbα-binding site in the A1 domain.

The C‐terminal acidic region in the A1 domain of factor VIII facilitates thrombin‐catalyzed activation and cleavage at Arg372

BackgroundFactor VIII (FVIII) is activated by thrombin‐catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin‐interactive sites responsible for cleavage at Arg372. We have also found that the A1 domain of FVIII bound to the anion‐binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337‐372 containing clustered acidic and hirugen‐like sequences. AimTo identify specific thrombin‐interactive site(s) within the A1 acidic region of FVIII. Methods and ResultsThe synthetic peptide of residues 337‐353 with sulfated Tyr346 (337‐353S) significantly blocked thrombin‐catalyzed FVIII activation and cleavage at Arg372, while a corresponding peptide of residues 354‐372 had no significant effect. Treatment with 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide to cross‐link thrombin and 340‐350S suggested that the 344‐349 clustered acidic region was involved in thrombin interaction. Alanine‐substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin‐catalyzed activation and cleavage at Arg372, with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin‐catalyzed activation and the cleavage rate at Arg372 using FVIII mutants with 337‐346 residues substituted with hirugen‐sequences (MKNNEEAEDY337‐346GDFEEIPEEY) were ~ 1.5‐ and ~ 2.5‐fold of WT, respectively. Surface plasmon resonance‐based analysis demonstrated that the Kd for active‐site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3‐ to 6‐fold higher than that of WT, and that the hirugen‐hybrid mutant facilitated association kinetics ~ 1.8‐fold of WT. ConclusionResidues 346‐349 with sulfated Tyr provided a thrombin‐interactive site responsible for activation and cleavage at Arg372. A hirugen‐hybrid A1 mutant showed more efficient thrombin‐catalyzed cleavage at Arg372.

Caplacizumab for Acute Thrombotic Thrombocytopenic Purpura.

Acute thrombotic thrombocytopenic purpura (aTTP) is a rare microangiopathic hemolytic anemia. Standard of care currently includes plasma exchange and immunosuppressive agents, including glucocorticoids, vincristine, and rituximab. Even with these therapies, relapse occurs in 36% of patients, and mortality ranges from 10% to 20%. Caplacizumab is a novel agent approved for the treatment of adult patients with aTTP in conjunction with plasma exchange and immunosuppressive therapies. It works by binding to the A1 domain of von Willebrand factor (VWF), blocking platelets from binding to VWF and aggregating. In clinical trials, patients who received caplacizumab compared with placebo were more likely to have a normalization of their platelet count, a lower rate of recurrence, and a lower incidence of the composite of aTTP-related death, recurrence, or major thromboembolic event. The side effect profile is rather benign and includes epistaxis, headache, and gingival bleeding. Caplacizumab is only available through specialty pharmacy services due to its high cost. Providers should be aware of and prepared for the prior authorization process required to assist their patients in gaining access to the medication. Currently, there is no formal consensus regarding caplacizumab's place in therapy for patients with aTTP, but it remains an option for refractory cases.

Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

The VWF/LRP4/αVβ3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signalling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

Structure of blood coagulation factor VIII in complex with an anti–C1 domain pathogenic antibody inhibitor

AbstractAntibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti–C1 domain inhibitor, termed 2A9, in complex with a B domain–deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti–C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

Conformation-dependent blockage of activated VWF improves outcomes of traumatic brain injury in mice

AbstractTraumatic brain injury-induced coagulopathy (TBI-IC) causes life-threatening secondary intracranial bleeding. Its pathogenesis differs mechanistically from that of coagulopathy arising from extracranial injuries and hemorrhagic shock, but it remains poorly understood. We report results of a study designed to test the hypothesis that von Willebrand factor (VWF) released during acute TBI is intrinsically hyperadhesive because its platelet-binding A1-domain is exposed and contributes to TBI-induced vascular leakage and consumptive coagulopathy. This hyperadhesive VWF can be selectively blocked by a VWF A2-domain protein to prevent TBI-IC and to improve neurological function with a minimal risk of bleeding. We demonstrated that A2 given through intraperitoneal injection or IV infusion reduced TBI-induced death by >50% and significantly improved the neurological function of C57BL/6J male mice subjected to severe lateral fluid percussion injury. A2 protected the endothelium from extracellular vesicle-induced injury, reducing TBI-induced platelet activation and microvesiculation, and preventing a TBI-induced hypercoagulable state. A2 achieved this therapeutic efficacy by specifically blocking the A1 domain exposed on the hyperadhesive VWF released during acute TBI. These results suggest that VWF plays a causal role in the development of TBI-IC and is a therapeutic target for this life-threatening complication of TBI.

Multimodal Imaging of Synaptic Vesicles with a Single Probe

A complete understanding of synaptic vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here we present a novel probe based on the membrane-binding C2 domain of cytosolic phospholipase A2 (cPLA2) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution.

Prioritization and characterization of validated biofilm blockers targeting glucosyltransferase C of Streptococcus mutans

To date, several Glucosyltransferase C (GtfC) inhibitors have been identified and experimentally validated. All these inhibitors have been validated at different experimental conditions like degree of purity, animal models, kinetic conditions, experimental environment etc.; and most of these inhibitors (ligands) proved to be quite effective in their respective validation environment. However, due to varied experimental validation conditions, and absence of molecular interaction data, there is no way to prioritize these validated ligands for their inhibition potential against GtfC. The present study is a novel attempt of comparative evaluation of the interaction of the validated ligands on a single platform and under similar conditions with a dual objective, i.e. ligand prioritization for their respective inhibitory potential and elucidation of the involved unknown molecular interactions. Carbohydrate derivatives (6-Deoxy sucrose and Trichloro-galactosucrose) were identified as the most promising GtfC inhibitors. In addition, Asp588, Trp517, and Asn481 amino acid residues of the domain A1 proved vital for the inhibitory effect. The study highlights the importance of the comparative analysis of the validated ligands in order to identify the most promising leads for drug discovery against dental caries.

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications.

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.

Hydrogen-Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.

CGTase, a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses.

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP‐10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP‐10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis‐related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens.

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

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

A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg372-Ser373, Arg740-Ser741, and Arg1689-Ser1690. Cleavage at Arg740 removes the B-domain, and cleavage at Arg1689 removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg372 separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg372 might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln372 backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIIIWT), FVIII372Q, and FVIII372Q followed by a rigid (Ala-Pro)5 linker sequence (FVIII372Q-AP5). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIIIWT, FVIII372Q and FVIII372Q-AP5 were completely resistant to cleavage at Gln372 by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIIIWT and FVIII372Q-AP5 displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIIIWT and FVIII372Q-AP5 displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII372Q was severely impaired. However, despite lack of cleavage at Gln372 in FVIII372Q-AP5, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIaWT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa372Q-AP5 was within a factor of 2 of that observed with FVIIIaWT while the rate observed with FVIIIa372Q was >10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa372Q-AP5(t1/2 ~ 10 min) compared to FVIIIaWT (t1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg372 for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa372Q-AP5 suggests that cleavage at Arg372 does not solely explain the spontaneous loss of FVIIIa cofactor function. Disclosures Krishnaswamy: Bayer: Research Funding.

Real-World Data with the Use of Caplacizumab in the Treatment of Acquired Thrombotic Thrombocytopenic Purpura (aTTP)

INTRODUCTION For more than two decades, the treatment of aTTPconsisted of therapeutic plasma exchange (TPE) and immunosuppressive agents. The addition of caplacizumab, a nano-antibody that binds to the A1 domain of the von Willebrand factor, inhibiting platelet aggregation, has been shown to reduce the time to resolution of thrombocytopenia, the rate of recurrence and the aTTP-related death. Real-world evidence of the effectiveness of caplacizumab is limited yet. The objective of our study was to assess the results of the introduction of caplacizumab in our internal protocol and to compare those results with the patients treated before the drug was available. METHODS A single-center retrospective observational study that evaluates the clinical characteristics and response to treatment of 18 consecutively diagnosed aTTP patients between May/14 to May/20. All patients received initial treatment with TPE and prednisone (PDN) 1 mg/Kg; the control group did not receive any other initial therapy, whereas nine patients received caplacizumab in addition to PEX and PDN once ADAMTS-13 deficiency was confirmed. Complete response (CR) was defined as the second of two consecutive days with platelets ≥150x109/L, refractoriness as the lack of platelet increase despite optimal therapy after 7 days, exacerbation as the decrease in platelet count during the first 30 days of discontinuation of TPE, and relapse as a new episode of aTTP beyond 30 days after the last TPE. All results are given as median (interquartile range). Statistical analysis was conducted using STATA/IC software. RESULTS The clinical characteristics at diagnosis of patients treated with or without caplacizumab were similar, except for a lower percentage of males and lower neurological involvement in the caplacizumab group (Table 1). Caplacizumab was started at a median of 3 days after diagnosis following ADAMTS-13 deficiency determination, and was administered during a median of 39 days (IQR 33-39). Adverse events related to caplacizumab were mild: 1 patient presented mild metrorrhagia, 1 developed pain and erythema at the puncture area and 1 suffered an urticarial dermatitis, the last case leading to the suspension of the drug since levels of ADAMTS-13 were recovered. The caplacizumab group achieved CR after a median of 4 days (IQR 3-4) vs. 6 days (IQR 5-14) in the control group (p = 0.016). Likewise, the number of TPE was lower with caplacizumab (Figure 1), with a median of 10 TPE (IQR 9-11) vs. 19 (IQR 16-23) (p = 0.001). Hospitalization time was also shorter in the caplacizumab group with a median of 12 days (IQR 12-14) vs. 26 (IQR 20-27) (p = 0.002). Finally the time of hospitalization into the intensive care unit was shorter in the caplacizumab group with a median of 3 days (IQR 2-4) vs. 4 (IQR 3-13) (p=0.1). In the caplacizumab group (median follow-up of 6.8 months), there were no refractory cases. There was 1 exacerbation before initiation of caplacizumab and 1 relapse. Both cases were treated with rituximab. In contrast, in the control group (median follow-up of 51.8 months), we observed 4 refractory cases (1 aTTP-related death), 3 exacerbations and 1 relapse; rituximab was necessary in 8 patients and a 3rd line with vincristine was administered in 4 cases. CONCLUSIONS The observed benefits of caplacizumab in our series are in line with the ones identified in randomized clinical trials. Caplacizumab can be used in combination with other therapies to attain a faster response and reduce aTTP-related complications. Disclosures Bosch: Hoffmann-La Roche: Research Funding.

Gene characterization and expression of the γδ T cell co-receptor WC1 in sheep

Sheep are known to express the hybrid co-receptor/pattern recognition receptor WC1 on their γδ T cells but details of the ovine WC1 multigenic array and gene expression were unknown. Annotation of the sheep genome assembly (Oar_rambouillet_v1.0) yielded 15 complete and 42 partial WC1 genes predicted to code for six different protein structures. RT-PCR amplification of the most distal scavenger receptor cysteine rich (SRCR) domain known as a1, which serves as the gene signature, from genomic and cDNA templates verified the majority of annotated genes. As for cattle and goats, sheep a1 domain sequences included WC1.1 and WC1.2 types. A unique ovine gene, WC1-16, had multiple SRCR a-pattern domains in tandem similar to one found in goats. Intracytoplasmic domains of WC1 transcripts had splice variants that may affect signal transduction. The larger number of WC1 genes in sheep and differences in structures and splice variants relative to cattle could have implications in expression patterns and engagement of γδ T cells by pathogens or vaccine constructs.

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.