Glycoprotein Research Articles

TANK-binding kinase 1 inhibitor GSK8612 suppresses platelet function and thrombosis via the ERK/P38 signaling pathway.

Inherited platelet function disorders (IPFD) are characterized by normal platelet count and morphology but impaired function due to pathogenic variants in genes encoding membrane receptors, granule constituents, or intracellular signaling proteins. Glanzmann’s thrombasthenia, the most representative IPFD, results from ITGA2B or ITGB3 mutations that disrupt the αIIbβ3 integrin complex, producing severe mucocutaneous bleeding. Advances in molecular genetics have expanded the IPFDs landscape to include defects in other platelet receptors (Glycoprotein (GP)-VI, P2Y12, and thromboxane A2[TxA2]-R), signaling mediators (RASGRP2, FERMT3, G-protein regulators, PLC, and TxA2 pathway enzymes), and granule biogenesis disorders such as Hermansky–Pudlak and Chediak–Higashi syndromes. High-throughput sequencing technologies, including long-read approaches, have greatly improved diagnostic yield and clarified genotype–phenotype correlations. Clinically, bleeding severity varies from mild to life-threatening, and management relies on antifibrinolytics, desmopressin, or platelet transfusion; recombinant activated factor VII and hematopoietic stem cell transplantation are reserved for selected cases. Emerging strategies such as gene therapy and bispecific antibodies that link platelets and coagulation factors represent promising advances toward targeted and preventive treatment. A better knowledge of the clinical features and understanding molecular pathogenesis of IPFDs not only enhances diagnostic precision and therapeutic options but also provides key insights into platelet biology, intracellular signaling, and the broader mechanisms of human hemostasis.

Ebola virus (EBOV) causes a severe human disease with high lethality. Pathogenesis of EBOV disease is characterized by a paradoxical combination of hyperinflammation and immunosuppression. EBOV has a single envelope glycoprotein (GP), which is a type I transmembrane protein with strong immunomodulatory effects. GP contains a conserved immunosuppressive domain (ISD) with a high similarity to ISDs of envelope proteins retroviruses. To investigate the effects of ISD, a set of 17 EBOV viral-like particle (VLP) constructs containing the entire EBOV nucleoprotein, VP40, and GP with single alanine or glycine substitutions in each position of ISD was generated and tested in human peripheral blood mononuclear cells (PBMCs). Wild-type VLPs induced inflammatory responses; however, when added to pre-stimulated cells, they reduced inflammation, thus exerting immunosuppressive properties. Substitution of lysine at ISD position 5 (Lys-5) increased anti-inflammatory properties by reducing proliferative responses of VLPs and also reducing nuclear factor of activated T-cells 1 (NFAT1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In contrast, substitution of tryptophan at position 14 (Trp-14) increased both the pro-inflammatory and the proliferative responses and the adhesion of VLP-infected human monocytes to microvascular endothelial cells. Thus, the ISD N- and C-termini have pro- and anti-inflammatory properties, respectively, suggesting their unique implications in the EBOV pathogenesis. Furthermore, the immunomodulating effects of ISD were also mediated by shed GP, which is abundant in the medium. These data may be useful for the development of treatments for diseases caused by EBOV by targeting the ISD.IMPORTANCEOur data suggest that the ISD N-terminus plays a role in activating immune cells and pro-inflammatory response. In contrast, the C-terminus of ISD downregulates the pro-inflammatory response through the reduction of NF-kB and NFAT activities. The data also show that EBOV GP increases the adhesion of monocytes to endothelial cells, and the effect is inhibited by the ISD C-terminus. Moreover, the data demonstrate that the immunomodulating effects of ISD are mediated not only by the virus-associated GP but also by the shed GP, which is abundant in the medium. Pathogenesis of the disease caused by EBOV is characterized by hyperinflammation and some features of immunosuppression, which could in part be affected by the complex effects of the ISD. These data indicate that targeting the ISD may be considered for the development of treatments for the disease caused by EBOV.

Patients with immune thrombocytopenia (ITP) remain a challenge to diagnose, manage and predict bleeding risk. A comprehensive assessment of platelet function may aid clinical management. This study assessed platelet parameters to predict bleeding in ITP. Blood from 103 clinically annotated cases with isolated thrombocytopenia and 123 healthy donors was evaluated. In the ITP cohort, 75/110 encounters (68%) had platelet counts below 50 × 109/L. Platelet surface proteins, reticulated platelets and activation were quantified using flow cytometry. Soluble receptor fragments, citrullinated histone-DNA (CitH3-DNA) complexes and thrombopoietin (TPO) were quantified by enzyme-linked immunosorbent assay (ELISA). Whole blood clotting and platelet contribution to clot formation were evaluated using viscoelastography. Elevated levels of glycoprotein (GP) VI (p = 0.0012), Trem-like transcript-1 (TLT-1) (p = 0.0248), platelet-bound immunoglobulin (Ig) G (p < 0.0029), CitH3-DNA complexes (p = 0.0022), TPO (p < 0.0001) and reduced platelet contribution to clot formation (p < 0.0001) were observed in primary ITP patients with bleeding and bruising symptoms. A multivariable analysis revealed that measuring platelet indices strengthened a predictive bleeding model over platelet count alone (78.1% vs. 70.48%). Symptomatic ITP patients have measurable platelet dysfunction and quantifiable differences on platelet surface, increased evidence of NETosis and elevated TPO levels. Identifying biomarkers for ITP outcomes can define subsets of disease with clinical relevance.

A minimum valency of 4 is required for robust activation of platelets in flow cytometry by multivalent nanobodies to Glycoprotein VI, C-type lectin-like receptor 2 and Platelet Endothelial Aggregation Receptor 1

Immune thrombocytopenia (ITP) is one of the most common causes of decreased platelet count. Bleeding is the main clinical symptom of ITP; although its severity correlates with the depth of thrombocytopenia, it may also depend on changes in the functional activity of platelets. In this study we have compared platelet functional activity in healthy volunteers (HV) and in ITP patients, as well as in groups of ITP patients with different levels of bleeding. The study included 65 HV and 84 ITP patients. Platelet activity was assessed by flow cytometry. Platelets were activated with thrombin receptor activating peptide (TRAP) or ADP, and the exposure of activation markers, activated form of glycoprotein (GP) IIb-IIIa and alpha-granule membrane protein P-selectin, was determined on their surface by measuring the binding of PAC-1 and CD62P antibodies, respectively. Platelet-associated IgG (PA-IgG, an indicator of the level of antiplatelet autoantibodies), the percentage of "young" reticular platelets (RP, %) and platelet light scatter (an indicator of their size) were also assessed using flow cytofluorimetry. Platelet binding of PAC-1 (and, to a lesser extent, CD62P binding) was lower in ITP patients than in HV. In ITP patients, PAC-1 binding inversely correlated with the PA-IgG content. In contrast to HV, in ITP patients, PAC-1 and CD62P binding did not directly correlate with the platelet size and RP, %. In ITP patients with severe bleeding, the platelet count was lower, PAC-1 and CD62P binding was reduced and PA-IgG and RP, % levels were increased. Thus, a decrease in the content of activation markers on the platelet surface was registered in ITP patients; it was more pronounced in patients with severe bleeding. It is suggested that the cause of this decrease may be due to the effect of autoantibodies (PA-IgG) on platelets, and in particular on GP IIb-IIIa.

Lassa virus (LASV) is circulating in rodents in several countries in West Africa and is the causative agent of the zoonotic disease Lassa fever. Several vaccine candidates have been successfully tested in preclinical and clinical research, while no LASV-specific vaccines or antiviral treatments have been licensed to date. Approximately 500,000 human cases of Lassa fever are estimated to occur every year. However, the high percentage (~80%) of asymptomatic cases and the low frequency of reporting systems in endemic regions demonstrate that Lassa fever cases are highly underreported. Given the frequent spread of the virus by travellers to non-endemic regions, the need for effective vaccines and treatments becomes clear. Here, we describe the generation and preclinical evaluation of two recombinant Lassa virus candidate vaccines, MVA-GP and MVA-NP, which are based on the highly attenuated modified vaccinia virus Ankara (MVA) strain. Constructed in the MVA vector, the MVA-GP vaccine delivers the glycoprotein (GP) of the prototype LASV Josiah strain (lineage IV), whereas the MVA-NP vaccine expresses the nucleoprotein (NP) from the Lassa virus Togo strain (lineage VII). Two immunizations of either MVA-GP or MVA-NP induced substantial polyfunctional Lassa virus-specific CD8+ and CD4+ T cell responses, respectively, in humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice (HLA-A*0201/DR1 transgenic mice). The identified human Lassa virus-specific T cell epitopes were in agreement with recently discovered T cell epitopes found in Lassa fever survivors. Further studies are warranted to characterize these recombinant MVA-Lassa virus vaccine candidates in other preclinical models and investigate their potential to be characterized in clinical studies in humans.

Marburg virus (MARV) infection can cause severe disease, and there is no available vaccine or therapeutic method. Research into potential vaccine design is focused on the glycoprotein (GP), which mediates the adherence and invasion process of the virus. However, it is unclear whether the degree of GP glycosylation is associated with vaccine efficacy. Here we constructed two versions of the GP expressed using insect and mammalian cell systems, respectively, either containing the mucin-like domain (MARV GPΔTM including residues 1-637) or deleting residues 264-425 to remove the part of mucin-like domain (MARV GPΔTM ΔMuc). Physicochemical properties, antigenicity, and immunogenicity were compared for soluble GPs produced in different cell expression systems. The GPΔTM ΔMuc produced in mammalian cells was more immunogenic, as evidenced by the induction of higher titers of binding antibodies and more antibodies targeting the protective epitope. Our results may offer a better understanding of glycosylation for the development of vaccines.

ABSTRACTFiloviruses pose a significant threat to human health due to frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. Here, we evaluate a general strategy for stabilizing glycoprotein (GP) structures from Ebola, Sudan, and Bundibugyo orthoebolaviruses and Ravn orthomarburgvirus. A 3.2 Å crystal structure provides atomic-level details of the redesigned Ebola virus GP, while cryo-electron microscopy reveals how a pan-orthoebolavirus neutralizing antibody targets a conserved site on the stabilized Sudan virus GP (3.13 Å resolution), along with a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N terminus to enable optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, we examine the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs in mice. Compared with the soluble trimer, SApNPs exhibit ∼112-fold longer retention in lymph node follicles, up to 28-fold greater presentation on follicular dendritic cell dendrites, and up to 3-fold stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wild-type and modified glycans are assessed in mice. This study provides a foundation for next-generation filovirus vaccine development.ONE-SENTENCE SUMMARYFilovirus glycoproteins and nanoparticles were rationally designed and characterized in vitro and in vivo to aid filovirus vaccine development.

Background and Aims: Evaluation of platelet morphology is a cornerstone of the diagnostic work-up of patients with suspected inherited platelet disorders (IPD). We have established an immunofluorescence microscopy-based method on the peripheral blood smear in combination with light-microscopy as a screening-tool for IPD. The technique has been validated for 9 disorders displaying typical changes of platelet structure. As a quality-control of this approach, we regularly perform a blinded investigation of subjects, who had previously undergone genetic testing. Methods: Blood smears were stained using 13 primary antibodies against different platelet structures (alpha and dense granule, surface glycoproteins, cytoskeletal components) and 2 fluorescence-labelled secondary antibodies. Exclusively based on the morphologic evaluation, we formulated a possible diagnosis. After uncovering patients’ information, we compared the anticipated defect with molecular outcome. Results: Over the last 12 months, we enrolled to this study 30 subjects. Based on the finding of platelet macrocytosis and reduced expression of the surface platelet glycoprotein (GP) Ib/IX, we predicted a diagnosis of Bernard Soulier syndrome (BSS) in 4 individuals. After unblinding molecular results, we could confirm the suspicion only in one case, in whom a pathogenic heterozygous deletion in GP1BB gene (c.236_244del [p.Pro79_Leu81del]) consistent with dominant-inherited BSS was found. In two subjects belonging to the same pedigree, a novel homozygous variant in the gene encoding for UDP-N-acetylglucosamine 2-epimerase (GNE): c.1516G&gt;A [p.Gly506Ser] was found. In one individual, we found two novel variants in compound heterozygosis in the gene encoding for uridine diphosphate (UDP)-galactose-4-epimerase (GALE): c.382G&gt;A [p.Val128Met] and c.590T&gt;C [p.Ile197Thr]. All the three patients suffered from severe thrombocytopenia. In the patient with GALE mutations, a syndromic picture including mental retardation, mitral valve prolapse and hip malformation was also apparent. Conclusions: Platelet macrocytosis with reduced expression of GPIb/IX can indicate inherited thrombocytopenias due mutations in GALE or GNE. These two genes play a role at different levels of platelet production and clearance. In both disorders, the reduced externalization of GPIb/IX is a consequence of impaired protein glycosylation. Immunofluorescence microscopy on the peripheral blood smear and genetic testing complement each other.

Structure-based identification of dual targeting lead inhibitor to Marburg virus glycoprotein and Chandipura virus nucleoprotein: Insights from molecular docking, dynamics and binding free energy analyses.

Vesicular stomatitis virus (VSV) pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (VSV-GP) represents a potent oncolytic virus (OV). Oncolytic virotherapy is an emerging anticancer approach that uses viruses to eliminate cancer cells by direct cell lysis and induction of an antitumor immune response. Immunomodulatory cargos expressed by OVs hold the potential to further enhance this antitumor immune response. To evaluate interleukin-12 (IL-12) as an immunomodulatory cargo encoded by VSV-GP, we used a subcutaneous tumor model by mixing type I interferon (IFN) competent murine lung epithelial cells (TC-1), which are largely resistant to VSV-GP in vivo, with VSV-GP permissive IFN-α receptor knockout TC-1 cells (TC-1ifnar1-/ -). This mixed model supports prolonged viral replication and subsequent IL-12 production. Oncolytic virotherapy with VSV-GP and VSV-GP-IL12 of parental TC-1 tumors did not lead to tumor control, whereas virus treatment in the TC-1/TC-1ifnar1-/- mixed tumors showed prolonged survival. Furthermore, VSV-GP-IL12 was even more effective than VSV-GP treatment. Analysis of CD8+ T cell responses revealed phenotypic differences of activated CD8+ T cells between VSV-GP and VSV-GP-IL-12 treatment, whereby VSV-GP-IL12-induced CD8+T cells displayed a phenotype described for long-lived effector cells (LLEC). Depletion experiments indicated that CD8+ T cells, and not NK cells, were responsible for the improved efficacy observed with VSV-GP-IL12 treatment. Taken together, we have demonstrated that oncolytic virotherapy using VSV-GP encoding IL-12 induces CD8+ T cell responses characterized by an LLEC phenotype, a cell population that is likely a crucial component of antitumor immunity.

Long-term antibody responses to the Ebola virus and the vaccine vector after rVSV-ZEBOV vaccination in DRC.

The rapid spread of infectious diseases presents a significant global threat, with seasonal influenza viruses, leading to 290,000–650,000 deaths annually. Emerging high pathogenic influenza strains from animals such as H5N1 and H7N9 further exacerbates pandemic risks. While developing effective vaccines and therapeutics is critical, the evaluation of these interventions is constrained by the requirement for high biosafety containment facilities. To circumvent these challenges, we developed S-Lux, a replication-deficient, single-cycle recombinant influenza virus expressing firefly luciferase (Flux) as a reporter protein. S-Lux can be pseudotyped with haemagglutinin from avian influenza, H5 and H7, enabling real-time monitoring of viral infection in vivo, and facilitate therapeutic antibody evaluation in low-containment facilities. In mice, S-Lux infection resulted in dose-dependent bioluminescent expression in the mouse airways and allowed evaluation of neutralising monoclonal antibodies and clearance of infected cells in mice. To extend this system, we generated ES-Lux by pseudotyping with the Ebola Glycoprotein (GP) and demonstrated that ES-Lux can be used to evaluate the efficacy of Ebola GP-targeting antibodies in vivo. Together, S-Lux and ES-Lux enable robust, simple and time-efficient assessment of antiviral therapy targeting influenza and Ebola virus in vivo, overcoming biosafety constraints that limit traditional efficacy studies.

Sialylated glycoproteins bind to Siglec-9 in a cis manner on platelets to suppress platelet activation.

Biomarkers of in vivo platelet activation in coronary artery disease: a systematic review and meta-analysis: communication from the SSC of the ISTH.

Comparison of EBOV GP IgG antibody reactivity: Results from two immunoassays in the Democratic Republic of the Congo.

The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1-GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1-GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1-GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1-GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1-GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1-GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.IMPORTANCEAlthough the emergence of New World (NW) hemorrhagic fever mammarenaviruses poses an unceasing threat to human health, there is a paucity of reagents capable of protecting against the transmission of these pathogens from their natural rodent reservoirs. This is, in part, attributed to our limited understanding of the structure and function of the NW glycoprotein spike complex presented on the NW arenavirus surface. Here, we provide a detailed molecular-level description of how the two major components of this key therapeutic target assemble to form a key building block of the NW arenaviral spike complex. The insights gleaned from this work provide a framework for guiding the structure-based development of NW arenaviral vaccines.

SummaryThrombin (FIIa) signaling through protease-activated receptors (PARs) is a relevant platelet activation mechanism. Human (h) PAR1 antagonists are approved for antithrombotic therapy, but bleeding is a concerning complication. In addition to PARs, platelet glycoprotein (GP) Ibα also binds FIIa enhancing human platelet response to lower agonist concentrations ex vivo. Signaling through GPCRs is well understood, but how GPIbα association with distinct PARs modulates FIIa-dependent platelet activation in vivo remains unclear. One obstacle in addressing this question is the distinct human platelet PAR1/PAR4 expression as opposed to mouse (m) PAR3/PAR4. Previous attempts to express functioning hPAR1 in mouse platelets using platelet-specific promoters or targeting into the mPAR3 locus have not been successful. Here we report our studies using a hPAR1 transgene with a floxed STOP sequence and strong synthetic (CAG) promoter targeted into the mouse Rosa26 locus. Generated Rosa26-hPAR1Tgfl mice were then sequentially crossbred with PF4–Cre and PAR3–/– mice, yielding mP3–/–hP1Tg mice whose platelets expressed hPAR1 with endogenous mPAR4. These mice, unlike PAR3–/–, had no excessive bleeding after tail clipping, but bled profusely after administration of the hPAR1 antagonist, vorapaxar. Accordingly, mP3–/–hP1Tg mice had more frequent and stable post injury occlusion of the carotid artery than PAR3–/– mice, but this difference was abolished by vorapaxar treatment. We anticipate that studies in this mouse strain will help unravel the regulation of PAR-mediated thrombin–induced platelet activation in vivo with findings more directly relevant to human pathophysiology.

A glycoprotein VI signaling defect in newly formed platelets generated in stress thrombopoiesis.