A1 Domain Research Articles

Identification of a VWFA1 Mutation Attenuating Thrombus Growth but Not Platelet Adhesion

The balance between hemostasis and thrombosis is critical to human health. However, intensified thrombosis that breaks this balance is often observed in diseases like hypertension, diabetes and obesity, leading to atherothrombosis in the coronary and cerebral circulations [1]. Current therapeutic strategies to reduce thrombotic risk are efficacious, as they have reduced morbidity and mortality, but still suboptimal, as commercialized drugs carry the risk of excessive bleeding. A new generation of drugs targeted to increase or maintain antithrombotic efficacy while reducing the negative impact on hemostasis is needed.The formation of platelet aggregates over an arterial vascular injury site is a two-step process. Firstly, binding between platelet glycoprotein (GP) Ibα - a component of the GPIb-IX-V receptor complex - and subendothelial von Willebrand Factor (VWF) at its A1 domain (VWFA1) enables recruitment of circulating platelets under high shear force. This represents the first layer of the hemostatic plug initiated by VWFA1-GPIb and consolidated by adhesion of activated integrins. Secondly, adhesion receptors on the up-facing side of the attached platelets engage circulating proteins including VWF, fibrinogen and fibronectin, among others, attracting new platelets into the growing thrombus. Importantly, this step is shared by atherothrombosis as a mechanism of thrombus growth. The GPIbα-VWFA1 interaction operates in both steps, which respectively mediate platelet adhesion and thrombogenesis [2]. Notably, when VWF is immobilized, its association with platelet GPIbα is mediated by two-dimensional (2D) kinetics; in contrast, the attachment of plasma VWF to immobilized platelets in the growing aggregate is governed by three-dimensional (3D) kinetics.We have identified positively charged, GPIbα-non-contact residues in helices α4/α5 of the VWFA1 that partially affect the VWF-GPIbα interaction. A triple mutation, R636/K642/K643A (referred to as M13) (Fig. 1A) drastically reduces the 3D on-rate of binding to GPIbα without significantly affecting the off-rate, which remains comparable to VWFA1-WT as measured by surface plasma resonance. In this assay, purified GPIbα molecules were immobilized on a surface that was then perfused with soluble monomeric VWFA1 at different concentrations. As a consequence of the decreased 3D on-rate, the 3D GPIbα binding affinity of M13 VWFA1 is 20-fold lower than that of WT VWFA1 (Fig. 1B). In agreement with this finding, aggregometry experiments showed that platelet agglutination mediated by dimeric M13 VWFA1 in the presence of ristocetin was greatly attenuated compared to WT (Fig. 1C), and the speed of agglutination was also slowed (Fig. 1D), implying that M13 is weaker in establishing platelet-platelet interactions.In contrast, the M13 mutation does not compromise the adhesion of platelets to immobilized VWFA1. In a flow chamber system, anticoagulated whole blood was perfused over a surface pre-coated with monomeric WT or M13 VWFA1. Under a range of shear rates and with different VWFA1 coating densities, the coverage of platelets over immobilized M13 VWFA1 was never less, if not greater than on WT VWFA1 (Fig. 1E,F, upper panels); accordingly, the rolling velocity of adherent platelets was similar or slower on M13 than WT VWFA1 (Fig. 1E,F, lower panels).We next investigated how the VWF M13 mutation affects in vivo thrombogenesis using a VWF-/- mouse strain in which platelets expresses human GPIbα. Mammalian cell expressed full-length human WT and M13 VWF was infused into these mice in combination with human FVIII to correct for the coagulation associated with VWFdeficiency; a carotid artery lesion was then caused with 9% ferric chloride. Our preliminary results showed that WT VWF restored a complete thrombotic occlusion of the artery, but mutant M13 VWF was without effect (Fig. 1G).Overall, our data suggests that VWFA1 domain can be engineered to support efficient platelet adhesion, which is key to hemostasis, while being less prone to mediate endovascular thrombogenesis. The development of this study should provide new insights into the distinct mechanisms that support VWF contributing to hemostasis or thrombosis, as well as into designing more selective pro-hemostatic VWF molecules with a reduced risk of causing thrombotic complications.

Characterization of a Nanobody That Binds Von Willebrand Factor and Prevents Von Willebrand Factor Cleavage By Plasmin

Introduction: Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adherence to exposed subendothelial collagen at sites of vascular injury. To prevent spontaneous platelet aggregation, accumulation of high molecular weight vWF multimers is prevented by ADAMTS13-mediated cleavage in the vWF A2 domain. Moreover, emerging data demonstrates plasmin-mediated cleavage of vWF in its open conformation in the A1-A2 linker, suggesting a role for plasmin in regulation of vWF multimer composition as a back up for ADAMTS13.Aim: We recently identified a llama-derived anti-vWF nanobody recognizing vWF in a conformation sensitive to plasmin cleavage. Our aim was to characterize this nanobody in terms of binding to- and interference with vWF functionality.Methods and results: The nanobody was produced by immunization of llamas with wildtype vWF, followed by preparation of cDNA from isolated peripheral blood lymphocyte-derived RNA and selection of the vWF A1 domain specific nanobody via phage display. To study recognition of vWF by the nanobody and possible modulating factors we developed an ELISA assay based on a nanobody-coated microtiter plate. Plasmin pre-treatment of normal pooled plasma (NPP) significantly increased nanobody recognition of vWF, but only after subjecting the plasma to shear by vortexing. Ristocetin also increased recognition by the nanobody; however, addition of plasmin to ristocetin-opened vWF in NPP did not further enhance nanobody binding. This suggests that, in contrast to ristocetin, vortexing was not sufficient to fully expose all vWF A1 domains, but the remaining hidden A1 domains were exposed by plasmin cleavage, allowing additional vWF to bind to the nanobody.Ellipsometry was used to assess competition of the nanobody and plasmin binding to vWF. Polyclonal anti-human vWF antibodies were coated to silicon slides, followed by addition of vWF concentrate (Haemate) or recombinant vWF R1306W, plasmin and the nanobody in varying order. We found that plasmin binds much more efficiently to R1306W vWF than to globular vWF. If the nanobody was bound to the R1306W vWF first, plasmin could not bind anymore. Conversely, after plasmin binding, only a small proportion of the nanobody still recognizes the plasmin-sensitive conformation of vWF, suggesting plasmin occupied the majority but not all binding sites and a possible difference in affinity for this conformation of vWF.A flow-cytometry based platelet-activation assay was performed to study the effect of plasmin on the ability of vWF in its plasmin-sensitive conformation to bind to washed platelets. Fluorescent detection of vWF binding was mediated by FITC-labeled monoclonal anti-vWF antibodies or biotin-conjugated labeled nanobody in combination with streptavidin-FITC. Addition of plasmin increased ristocetin-modified vWF binding to platelets by 3.7-fold. This effect of plasmin was abrogated in the presence of heparin, consistent with work by Brophy et al. (2017) showing that heparin prevents cleavage of vWF by plasmin. Hence, it appears that plasmin-cleaved vWF has increased binding affinity to platelets. Of note, a similar pattern in ristocetin-activated vWF to platelet binding upon addition of plasmin (and heparin) was detected by the nanobody. This demonstrates that the nanobody does not interfere with platelet binding of vWF. Moreover, binding of plasmin-cleaved vWF resulted in reduced platelet activation, as determined from P-selectin expression.Discussion: In conclusion, we have identified an anti-vWF nanobody capable of blocking plasmin binding to- and subsequent cleavage of vWF. This nanobody is unable to bind globular vWF in solution or vWF immobilized by polyclonal anti-vWF antibodies, but efficiently interacts with shear- and ristocetin-activated vWF and recombinant vWF comprising the type 2B R1306W mutation. The nanobody epitope on vWF is expected to overlap with- or lie in close proximity to the plasmin binding site in the A1-A2 linker, possibly resulting in steric hindrance. Current studies focus on providing more direct evidence of the vWF binding site of the nanobody. From a clinical perspective, the sensitivity of R1306W vWF to plasmin-mediated cleavage may explain the bleeding diathesis in VWD type 2B patients with this mutation. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand Factor A1 Domain Binds to Clusters II and IV of Low-Density Lipoprotein Receptor-Related Protein 1

Introduction. Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein involved in hemostasis and thrombosis. Genetic and in vivo studies have identified low-density lipoprotein receptor-related protein 1 (LRP) as the major clearance receptor of VWF. The ligand-binding moiety of LRP is represented by complement-type repeats grouped into four clusters, among which clusters II and IV are prominent in ligand binding. In in vitro studies under shear stress conditions, VWF acquires an unfolded conformation and interacts with LRP. More than 30 mutations of VWF have been reported to associate with its increased plasma clearance and result in type 2B von Willebrand disease. The majority of these mutations are located within the VWF A1 domain (A1), which is in agreement with previous reports that isolated A1 binds to LRP. However, the interactive site of LRP has not been identified.Objective. We aim to characterize the LRP binding site for VWF by investigating the interaction of individual LRP clusters II-IV to different A1 variants.Experimental Approach. An A1-containing large N-terminal homo-dimeric proteolytic fragment of VWF (SPIII) was produced upon its proteolytic digestion by V8-protease. Alternatively, we generated three variants of recombinant A1: (i) A1(residues 1261-1478), (ii) a VWD type 2B mutant, A1(V1316M) (residues 1261-1478) and (iii) an N-terminus truncated form, (residues 1269-1478). These proteins were tested for binding to immobilized LRP, purified from human placenta, and recombinant LRP clusters II and IV by surface plasmon resonance.Results. We did not find any interaction between the SPIII proteolytic fragment and LRP, suggesting that this fragment may still preserve a folded conformation of VWF that masked its LRP-binding site. In turn, all three forms of recombinant A1 were found to bind LRP and its clusters II and IV. Notably, A1(V1316M) and A1(1269-1478) had higher affinity for LRP and its clusters than A1. This result is consistent with the enhanced clearance of the VWF(V1316M) in vivo and better exposure of the A1 site in a truncated form of A1 to the platelet receptor GpIb-alpha reported previously.Conclusions. Our data demonstrate that LRP has at least two sites for binding to VWF, which are located within clusters II and IV. This finding is in accordance with previous observations that LRP clusters II and IV can serve as “functional duplicates” for binding selected ligands. DisclosuresNo relevant conflicts of interest to declare.

Influence of Inhibitor Characteristics on Response to Hemostatic and Immunosuppressive Treaments in Acquired Hemophilia Patients: The Ciha Project

INTRODUCTIONAcquired hemophilia (AH) is a disease induced by autoantibodies targeting coagulation factors, mostly factor VIII (FVIII). The disease manifests as bleeding episodes in unusual locations that usually prompt physicians to establish hemostatic and immunosuppressive treatments (IST) for hemorrhage cessation and inhibitor eradication respectively. The specificity of the so-called inhibitors varies among patients. We present herein the main findings of the CIHA project, which is a pilot study aimed to test the hypothesis that the inhibitor's features, i.e., its FVIII domain specificity, influences AH patient evolution and response to hemostatic and immunosuppressive therapies.METHODSThis is a prospective, descriptive, observational and non-interventional study. Between September 2011 and September 2013, 16 patients who had been diagnosed with AH caused by FVIII inhibitors were recruited, and their clinical and epidemiologic features were assessed. Inhibitors were titrated by the Bethesda method according to the Nijmegen modification. The inhibitor specificities were determined by Western blot: first, FVIII was cleaved by thombin in order to obtain the fragments corresponding to the light chain (LC), and the A1 and the A2 domains of the heavy chain. Once electrophoresed and transferred to a polyvinylidine difluoride membrane, this material was incubated with the patient´s plasma. Finally, an anti-human IgG antibody carrying a chemiluminiscent substrate was incorporated. The luminiscent pattern should be dependent on the molecular weight of the FVIII domains recognized by the inhibitor, allowing us to assess the inhibitor's specificity. In order to analyze the association between inhibitors' specificities and individual responses to therapies, sampling was performed at the time of diagnosis, 14 days afterwards, and, provided that remission was reached, 56 days after IST was withdrawn. When immunosuppression failed, a similar sampling pattern was applied after a second line of therapy was initiated.RESULTSThree patients died in the course of the study due to complications linked to their underlying pathologies related neither to the hemostatic treatment nor to the IST.According to the literature, inhibitors targeted mostly the LC (11 out of 16; 69%) or the A2 domain (8 out of 16; 50%). Two of them (12%) were directed against the A1 domain. It must be remarked that, in disagreement with the literature, the inhibitors targeted more than one domain in a non-negligible number of cases [5 out of 16 (31%): A2-LC, three cases; A1-A2-CL and A1-A2, one case each].Bypass agents, i.e., recombinant activated factor VII or activated prothrombin complex, were used for hemorrhage cessation. The inhibitor features did not influence the response to hemostatic treatment, since this efficiently stopped blood loss in all patients.The first line immunosuppressive drugs most commonly used were steroids (14 out of 16), alone or with concomitant cyclophosphamide (6 out of 14). Nine patients (56%) reached remission, and the rest of them required a second IST to eradicate the inhibitor. Interestingly, inhibitors targeted LC in 6 out of 7 (86%) patients that did not respond to the first IST. Conversely, inhibitors targeted LC only in 4 out of 9 (44%) patients who reached remission with the first line IST. Finally, 30 days after IST had started, inhibitor specificity changed in one of the IST responders, from multidomain targeting (A1-A2-LC) to LC-only targeting.CONCLUSIONSAntibody recognition of more than one FVIII domain is not uncommon in AH. More importantly, this pilot study suggests that inhibitors that interact with the LC of FVIII may induce a worse response to IST in AH patients. Since this finding may be clinically relevant, these results encourage us to carry out a larger study to confirm such observations. DisclosuresNo relevant conflicts of interest to declare.

FVIII Proteins with a Modified Immunodominant T-Cell Epitope Exhibit Reduced Immunogenicity and Normal Procoagulant Activity

Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients. DisclosuresEttinger:Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James:Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt:Bloodworks NW: Patents & Royalties: inventor on FVIII patents.

Characterization of the HLA-Class-II (HLAcII)/Therapeutic Factor VIII (FVIII)-Derived Peptidome By FVIII Domain and HLAcII Isomer

Abstract Background: Hemophilia A (HA) is caused by F8 mutations and resultant plasma deficiencies in FVIII activity. While bleeding can be arrested by infusing therapeutic FVIII proteins (tFVIIIs), ~30% of severe HA patients will develop neutralizing tFVIII antibodies (inhibitors) and become resistant to treatment. For inhibitors to develop, foreign peptides must be liberated from a tFVIII and displayed on dendritic cells (DCs) in self HLAcII molecules. Little is known about the inhibitor risk attributable to distinct isomers of individual HLAcII repertoires and different tFVIII domains. Aims: Determine the contributions to FVIII immunogenicity risk of the distinct 1) HLAcII isomers DP, DQ and DR, and 2) tFVIII domains A1, A2, A3, B, C1 and C2. Methods: We analyzed HLAcII/tFVIII peptidomic data generated in DC-protein processing assays (PPAs) that used monocyte-derived DCs from 12 healthy donors to compare the immunogenicity of a full length (FL) and B-domain truncated (BDT) recombinant FVIII (FL- and BDT-FVIII) ± von Willebrand Factor (VWF) and a plasma derived (pd)FVIII + VWF. All tFVIIIs contain 3 small acidic-residue-rich peptides (a1, a2 and a3) that connect A1 to A2, A2 to B, and B to A3. To simplify our analysis, any LC-MS/MS individually sequenced peptide (ISP) with an N-terminal residue in either the (i) C-terminal portion of A1, A2 or B that extends into the adjacent a1, a2 or a3 region, respectively, was placed into that domain, or (ii) a1, a2 or a3 region, whether or not it extends into A3, C1 or C2, was placed into that domain. We then calculated the expected frequency of peptides from each FVIII domain (including, when appropriate, the adjacent acidic regions) based on residue number. Finally, we compared the observed and expected peptide proportions and performed a Z-test on the difference of proportions per domain. We performed this analysis for all ISP data in which the observed frequencies were obtained by a weighting scheme jointly taking into account contributions by tFVIII & by domain (All-data). We also performed this analysis by tFVIII. Results: For the All-data comparisons, we found that A3 had a higher observed than expected proportion (dP = 22%; p = 0.006). For the FL-FVIII comparisons, the B domain had a lower observed than expected proportion (dP = -12.1%; p We also approached these data from the HLAcII perspective. We found that our 12 donors expressed 12 distinct DR molecules based on their DRB1 alleles. After tallying the per allele density of FVIII residues (for all 2332 residues) falling in the DR-bound fraction of peptides, we constructed FVIII residue-by-allele matrices of dimensions 2332 x 12 per tFVIII and performed singular value decompositions that yielded their respective matrices of right singular vectors also known as principal components (PCs). Such PCs are said to span or generate a corresponding vector subspace. Using matrix analytic methods, we can compare the therapeutic-specific subspaces to see if such contrasts reflect differences in their associated density profiles. To compare two subspaces, we compute their projection onto their intersection subspace, and use this projection to compute a subspace distance. We test the null hypothesis of a subspace distance equal to 0 (for statistically similar subspaces) by a permutation test. As an example, we compared the FL-FVIII + VWF and pdFVIII + VWF subspaces, and found that they are significantly different (subspace distance (SD) = 0.96; p Conclusions: Our findings demonstrate that the distinct domains of tFVIIIs and different alleles of individual HLAcII repertoires differentially contribute to inhibitor risk. Download : Download high-res image (175KB) Download : Download full-size image Disclosures Hofmann: CSL Behring: Employment. Escobar: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinh: Haplomics: Employment. Powell: CSL Behring: Employment. Howard: CSL Behring: Honoraria, Research Funding; Haplomics: Employment, Equity Ownership, Patents & Royalties, Research Funding.

PtFVa (Pseudonaja Textilis Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C.

OBJECTIVE: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). APPROACH AND RESULTS: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. CONCLUSIONS: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.

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

The GPIbα-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 (GpIbaΔsig/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 von Willebrand factor (VWF) normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 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 collagenrelated- peptide (CRP) exhibited markedly decreased P-selectin exposure and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in GPVI-mediated signaling. Consistent with this, while hemostasis 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 aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.

High-resolution structure of the vWF A1 domain in complex with caplacizumab, the first nanobody-based medicine for treating acquired TTP

von Willebrand factor (vWF) is a huge oligomeric glycoprotein involved in blood homeostasis. However, this protein is also implicated in acquired thrombotic thrombocytopenic purpura (TTP). The blocking of its binding with platelets has been recognized as an attractive therapeutic strategy for treating acquired TTP. Caplacizumab, a bivalent single-domain antibody (VHH), is the first FDA-approved nanobody drug against vWF for the treatment of acquired TTP. Here, we describe the crystal structure of the A1 domain of vWF in complex with the caplacizumab nanobody at the resolution of 1.60 Å. This structure elucidates the precise epitope and binding mode of caplacizumab. Unexpectedly, caplacizumab binds to the bottom face of the vWF A1 domain and does not create any steric clash with platelet-receptor glycoprotein Ib (GPIb) bound to vWF. However, its binding can stabilize the different conformation within the N-terminus and α1β2 loop from the GPIb bound structure, suggesting that the mechanisms of caplacizumab would not be the direct competition of GPIb binding to vWF A1 domain but the conformational arrestment of vWF in an inappropriate state to platelet adhesion. This high-resolution structure would provide helpful information for the design of improved anti-vWF therapeutics for the treatment of acquired TTP.

GFAP variants leading to infantile Alexander disease: Phenotype and genotype analysis of 135 cases and report of a de novo variant

ObjectivesAlexander disease (AxD) is a rare autosomal dominant disorder due to GFAP mutations; infantile AxD is the most common severe form which usually results in death. In this study, phenotype and genotype analysis of all reported cases with IAxD are reported as well as a de novo variant. MethodsWe conduct a comprehensive review on all reported Infantile AxD due to GFAP mutation. Clinical data and genetics of the reported patients were analyzed. Clinical evaluations, pedigree drawing, MRI and sequencing of GFAP were performed. Results135 patients clinically diagnosed with IAxD had GFAP mutations. A total of fifty three variants of GFAP were determined; 19 of them were located at 1A domain. The four common prevalent variants (c 0.715C>T, c 0.236G˃A, c 0.716G˃A, and c 0.235C˃T) were responsible for 64/135 (47.4%) of the patients. Seizure was the dominant clinical symptom (62.3%) followed by macrocephaly (41%), developmental delay (23.9%) and spasticity (23.9%). A de novo variant c 0.715C˃T was found in the presented Iranian case. DiscussionThe majority of GFAP variant are located in a specific domain of the protein. Seizure as the most common symptom of IAxD could be considered. This study highlighted the role of genetic testing for diagnosing AxD.

The Intracellular N-Terminal Domain of the Acid-Sensing Ion Channel 1a Participates in Channel Opening and Membrane Expression.

Acid-sensing ion channels (ASICs) are widely expressed in the nervous system. The intracellular C terminus of ASIC1a has many sites involved in regulating its expression and the opening mechanism, but the role of the intracellular N-terminal domain is poorly understood. Here, we explored the correlation of ASIC1a intracellular N terminus with membrane expression and gate opening. We modified the N-terminal structure of ASICs by deletion/truncation/mutation strategies and transfected the recombinant plasmids into CHO cells. Protein expression was analyzed with immunofluorescence, Western blots, and patch-clamp experiments. Deleting the entire N terminus decreased the membrane expression of channel proteins, and ion channel opening was lost. Deleting sections of the N terminus also decreased membrane expression and suggested that all areas were significant, with no single or group of amino acid residues playing a decisive role in regulating ASIC1a membrane expression. In terms of gate opening, five amino acid (AA) residues from AA 16 to AA 20 participated in gate opening, and isoleucine at AA 18 was the most important. The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid residues (AA 16-20) are involved in the gate opening mechanism. SIGNIFICANCE STATEMENT: The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid resi-dues (amino acid 16-20) are involved in the gate opening mechanism.

Contrast-enhanced ultrasound molecular imaging of activated platelets in the progression of atherosclerosis using microbubbles bearing the von Willebrand factor A1 domain.

Platelet-endothelial interactions have been linked to increased inflammatory activation and a prothrombotic state in atherosclerosis. The interaction between von Willebrand factor (vWF)-A1 domain and platelet glycoprotein (GP) Ib/IX plays a significant role in mediating the adhesion of platelets to the injured endothelium. In the present study, contrast-enhanced ultrasound (CEU) molecular imaging with microbubbles bearing the vWF-A1 domain was performed to non-invasively monitor activated platelets on the vascular endothelium in the procession of atherosclerosis. A targeted CEU contrast agent was prepared by attaching the vWF-A1 domain to the shell of microbubbles (MbA1). Rat isotype control antibody was used to produce control (Mbctrl) microbubbles. The binding of MbA1 and Mbctrl to activated platelets was assessed in in vitro flow chamber experiments. Apolipoprotein E (ApoE-/-) deficient mice were studied as a model of atherosclerosis. At 8, 16 and 32 weeks of age, CEU molecular imaging of the proximal aorta with MbA1 and Mbctrl was performed and the imaging signals from microbubbles were quantified. Atherosclerotic lesion severity and platelets on the endothelial surface were assessed by histology and immunohistochemistry. In in vitro flow chamber studies, attachment of MbA1 to activated platelets on culture dishes was significantly greater than that of Mbctrl across a range of shear stresses (P<0.05). The attachment of Mbctrl was sparse and not related to the aggregated platelets. As lesion development progressed in the ApoE-/- mice, molecular imaging of activated platelets demonstrated selective signal enhancement of MbA1 (P<0.05 vs. Mbctrl) at all ages. Selective signal enhancement from MbA1 increased from 8 to 32 weeks of age. Immunohistochemistry for GPIIb revealed the presence of platelets on the endothelial cell surface in each group of ApoE-/- mice and that the degree of platelet deposits was age-dependent. The results of the present study indicated that non-invasive CEU molecular imaging with targeted microbubbles bearing the vWF-A1 domain could not only detect activated platelets on the vascular endothelium but also indicate lesion severity in atherosclerosis.

The Activity of von Willebrand Factor in Hospitalized Patients with COVID‐19

Infection with the SARS‐CoV‐2 virus, or COVID‐19, is associated with an increased risk of thrombosis at all levels of the vascular tree including the microvasculature, arteries and veins. However, the mechanisms involved in the heterogenic thrombotic presentations in COVID‐19 are poorly understood. One potential contributor to the prothrombotic state in COVID‐19 is von Willebrand Factor (vWF), a multimeric glycoprotein produced by endothelial cells and platelets that is unfolded and activated by shear stress or artificially with ristocetin. vWF has been implicated in pathogenesis of arterial clots including stroke, consistent with its activation by shear. vWF has also been implicated in thrombotic thrombocytopenic purpura, a subtype of microvascular thrombosis. Elevated vWF has even been associated with increased risk of venous thrombosis, a classically low shear environment. The purpose of this study was to determine whether vWF had a higher propensity to become activated in COVID‐19, suggestive of an altered configuration. We hypothesized that vWF from patients with COVID‐19 would be more active than healthy controls. Discarded whole blood samples were collected from hospitalized patients diagnosed with COVID‐19; blood from healthy donors was collected via venipuncture. Patient and control plasma were rapidly thawed at 37 ℃ then transferred to de‐identified containers to blind performing scientist. Samples were processed in a duplicate manner adding 30 μL of plasma to a solution containing 200 x103/μL lyophilized platelets. We measured vWF activation using two methods: the first involved agglutination with an intermediate concentration of ristocetin (between 0.6 ‐ 0.7 mg/mL) to achieve less than 40% of normal agglutination. The second method involved detection of A1‐12 antibody binding with ELISA. The A1‐12 antibody binds to the N‐terminus of the A1 subunit, a location that is typically obscured by the A2 domain and thus is only accessible with vWF is unfolded. All measured values were compared to normal pooled plasma (NPP) processed in the same experiment. Notably all measures of activation of vWF were significantly higher in COVID‐19 patients compared to healthy controls. The primary slope of the agglutination was the most significantly elevated when compared to controls (162 verses 106 % of NPP, p = 0.002). The absolute amount of vWF was also significantly increased in patients compared to controls (148 verses 100% of NPP, p < 0.001). These preliminary data are suggestive of increased activity of vWF in COVID‐19 infectionin addition to an increased absolute level. vWF may be a significant pathway that becomes dysregulated during the immune response to SARS‐CoV‐2. This study is limited by its use of discarded blood that has undergone one freeze‐thaw cycle. Further work is needed to correlate these results to clinical outcomes.

Mucosal LTA1 Elicits Lung Th17 cells with reduced plasticity towards Th1 cells and resistance to FK506.

Abstract Objective: Based on our previous work, we identified a vaccine composed of the A1 domain of heat-labile toxin from E. coli (LTA1) adjuvant and Outer membrane protein (Omp) X from K2 strain of Klebsiella pneumoniae (K. pneumoniae), which can elicit serotype independent protection against K1 strain by generating lung Th1 and Th17 cells. To better understand the characteristics of these Th17 cells, we investigated the TCR repertoire, plasticity, and resistance to FK506. Methods: Naïve or immunized lung cells were collected and stained with Anti-Mouse TCR Vβ Screening Panel for FACS. Naïve CD4+ cells were isolated from spleens and cultured under Th17 differentiation conditions in vitro. Immunized lung Th17 cells were purified. To assess plasticity towards Th1, cells were cultured w/wo IL-12 and analyzed by FACS. To assess resistance to calcineurin inhibitors, the naïve/immunized CD4+ cells were treated w/wo FK506 both in vivo and ex vivo, analyzed by FACS. Results and Conclusions: The LTA1-OmpX elicited CD4+ cells were oligoclonal with skewing towards TCRVβ14+ cells. After incubation with IL-12, 57.73 ± 3.9% of the in vitro differentiated Th17 cells showed plasticity towards Th1 cells whereas in immunized lung Th17 cells only showed 6.25 ± 1.6%. Lung Th17 cells also showed substantially reduced STAT4 phosphorylation and surface IL-12rβ2 expression compared to in vitro Th17 cells. Furthermore, lung Th17 cell IL-17A expression was resistant to FK506 treatment both in vivo and ex vivo, which potentially indicated that this vaccine strategy may confer protection in the setting of transplant immunosuppression.

Clinical spectrum and genetic variations of LMNA-related muscular dystrophies in a large cohort of Chinese patients

BackgroundLMNA-related muscular dystrophy is caused by mutations in LMNA gene. We aimed to identify genetic variations and clinical features in a large cohort of Chinese patients with LMNA mutations in...

Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module

Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

BackgroundVon Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous...

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.

Novel splicing (c.6529-1G&gt;T) and missense (c.1667G&gt;A) mutations causing factor V deficiency

Congenital factor V deficiency (FVD) is a rare bleeding disorder. In this study, we investigated the genetic basis in an African American patient with factor V activity 3%. Custom sequence capture and targeted next-generation (NGS) sequencing of the F5 gene were undertaken followed by PCR and Sanger sequencing. Two novel variants were identified. In silico analyses correlated clinically with the patient's factor V activity and hemorrhagic tendency. A review of the literature regarding these genomic alterations is presented. We described two novel mutations causing moderate FVD. The first, Chr1:g.169483698C>A with cDNA change (F5):c.6529-1G>T, occurred in a conserved nucleotide at the canonical acceptor splice site of intron 24. The second, Chr1:g.169515775C>T with cDNA change (F5):c.1667G>A, was a missense variant of exon 11, affecting a highly conserved amino acid in the A2 domain. Further research into the mechanisms of F5 mutations leading to FVD and residual factor V expression are needed.