Glycoprotein Ibα inhibitor (CCP-224) prevents neutrophil-platelet aggregation in Sickle Cell Disease.
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ABSTRACT:
SUBMITTER: Jimenez MA
PROVIDER: S-EPMC5617353 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
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Glycoprotein Ibα inhibitor (CCP-224) prevents neutrophil-platelet aggregation in Sickle Cell Disease.
Jimenez Maritza A MA Novelli Enrico E Shaw Gray D GD Sundd Prithu P
Blood advances 20170901 20
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Project description:BackgroundPlatelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions.MethodsBioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions.ResultsDeletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage.ConclusionsOur results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.
| S-EPMC6464389 | biostudies-literature
Project description:Background and purposePlatelet membrane glycoprotein Ibα (GPIbα), the major ligand-binding subunit of the GPIb-IX-V complex, binds to a number of ligands contributing to hemostasis, thrombosis, and inflammation. Binding to von Willebrand factor (VWF) initiates the process of hemostasis/thrombosis, while binding to the leukocyte receptor Macrophage-1 antigen (Mac-1) has been implicated in modulating the inflammatory response. Thus as GPIbα resides at the nexus of thrombosis and inflammation, we investigated the impact of GPIbα on radiation injury outcomes as this injury triggers both the thrombotic and inflammatory pathways.Materials and methodsWe used wild-type (WT) C57BL/6J mice and a dysfunctional GPIbα mouse model, in which endogenous GPIbα is replaced with a non-functional α-subunit (hIL-4R/Ibα), to determine whether the impairment of platelet GPIbα alters radiation response. Following exposure to 8.5 Gy total body irradiation (TBI), a series of parameters including radiation lethality, platelet-neutrophil/monocyte interactions, neutrophil/monocyte activation, serum cytokine levels and intestinal injury, were compared between the strains.ResultsThe lack of functional GPIbα resulted in higher radiation lethality, greater monocyte activation, increased levels of serum pro-inflammatory cytokines, heightened intestinal damage, and a reduction of intestinal neutrophil recovery.ConclusionThese data suggest that loss of platelet GPIbα enhances radiation toxicity and that GPIbα-mediated interactions may play a crucial role in limiting radiation damage. Thus, a mechanistic understanding of the biological impact of GPIbα following TBI could provide crucial insights for improving the safety of radiotherapy and minimizing the deleterious effects of accidental or occupational exposure to high-dose radiation.
| S-EPMC8934272 | biostudies-literature
Project description:BackgroundPlatelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized.ObjectivesThe aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures.MethodsGPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans.ResultsWe identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain.ConclusionsThis comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.
| S-EPMC10065957 | biostudies-literature
Project description:The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.
| S-EPMC8876638 | biostudies-literature
Project description:Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections. Using a mouse model of S. aureus-induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection.
| S-EPMC11546206 | biostudies-literature
Project description:Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates via the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). In vivo, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic in vitro system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such in vitro studies can be useful to determine optimum therapeutic ranges for targeting this interaction.
| S-EPMC4122703 | biostudies-literature
Project description:Background: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. Objectives: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. Methods: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans. Results: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified at least 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD). Conclusions: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.
2023-02-09 | PXD035030 | Pride
Project description:The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser(609) of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca(2+) levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.
| S-EPMC3382782 | biostudies-literature
Project description:BackgroundStoring platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor.Design and methodsWe examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy.ResultsCold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-D-glucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions.ConclusionsWe conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.
| S-EPMC3590094 | biostudies-literature
Project description:During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets.
| S-EPMC7776245 | biostudies-literature