Comparative study of the glycosylation of platelet glycoprotein GPIIb/IIIa and the vitronectin receptor. Differential processing of their beta-subunit.
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ABSTRACT:
SUBMITTER: Troesch A
PROVIDER: S-EPMC1131401 | biostudies-other | 1990 May
REPOSITORIES: biostudies-other
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Project description:The interaction of human thrombospondin (TSP) with GPIa-IIa and GPIIb-IIIa was studied. The binding for both proteins became time-independent after 60 min. A 7-fold excess concentration of unlabelled GPIa-IIa added either initially, or after time-dependent binding, resulted in a 50% inhibition of GPIa-IIa bound to TSP. GPIa-IIa and GPIIb-IIIa specifically bound TSP since: (a) the binding of GPIIb-IIIa to TSP was dependent on the presence of 1 mM MgCl2 and 1 mM CaCl2, whereas binding of GPIa-IIa was ion-independent. (b) The binding was saturable, with dissociation constants of 0.69 +/- 0.17 microM and 3.77 +/- 1.02 microM for GPIa-IIa and GPIIb-IIIa respectively. (c) GPIIb-IIIa and GPIa-IIa did not significantly bind to BSA. (d) GPIIb-IIIa bound fibrinogen ion-specifically, whereas little or no binding of GPIa-IIa was detectable. (e) Both GPIIb-IIIa and GPIa-IIa bound collagen in an ion-independent manner. (f) GPIIb-IIIa did not compete with GPIa-IIa for binding to TSP. (g) Binding of GPIa-IIa to TSP was inhibited with anti-(GPIa-IIa) (6F1), whereas mouse IgG and anti-(GPIIb-IIIa) (AP-2) had no effect. (h) The interaction of GPIa-IIa with TSP is 5.5-fold more favourable than that of GPIIb-IIIa suggesting that GPIa-IIa may be a preferred binding protein for TSP-mediated platelet adhesion.
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Project description:ObjectivesPlatelet activation and aggregation play an important role in the pathogenesis of cardiovascular disease. We examined the association of a single nucleotide polymorphism (SNP) in the GPIIIa platelet glycoprotein (Leu33Pro) with carotid artery plaque morphology and with expression of platelet markers using data from the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study.MethodsThe study sample consisted of 1202 Caucasian members of the ARIC study cohort recruited in 2004-2005 to participate in the Carotid MRI Substudy under stratified sampling based on maximum carotid artery wall thickness. The Leu33Pro polymorphism was identified as SNP rs5918 in the ITGB3 gene. Plaque visualization was accomplished with contrast enhanced MRI examination of the thickest segment of the carotid artery. Expression of platelet markers was measured using fasting whole blood flow cytometry.ResultsThis cross-sectional analysis based on age and gender adjusted weighted linear regression models suggests that those homozygous for the Leu33Pro risk allele (C) have decreased mean and minimum fibrous cap thickness. We did not observe differences in plaque lipid volume or maximum carotid artery wall thickness across SNP rs5918 genotypes. Carriers of the Leu33Pro polymorphism, as compared to major allele homozygotes, had greater percent of platelets expressing P-selectin, a platelet glycoprotein indicating activation status. Prevalent coronary heart disease did not affect estimates of fibrous cap thickness or of platelet activation.ConclusionOur results suggest that individuals with Leu33Pro polymorphism of the GPIIIa glycoprotein may be predisposed to increased risk of atherosclerotic plaque rupture.
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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.
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Project description:In ovarian cancer, ascites represent the microenvironment in which the platelets extravasate to play their role in the disease progression. We aimed to develop an assay to measure ascites' platelet activation. We enriched small extracellular vesicles (EVs) (40-200 nm) from ascites of high-grade epithelial ovarian cancer patients (n = 12) using precipitation with polyethylene glycol, and we conducted single-particle phenotyping analysis by nano-flow cytometry after labelling and ultra-centrifugation. Atomic force microscopy single-particle nanomechanical analysis showed heterogeneous distributions in the size of the precipitated particles and their mechanical stiffness. Samples were fluorescently labelled with antibodies specific to the platelet markers GPIIb/IIIa and PF4, showing 2.6 to 18.16% of all particles stained positive for the biomarkers and, simultaneously, the EV membrane labelling. Single-particle phenotyping analysis allowed us to quantify the total number of non-EV particles, the number of small-EVs and the number of platelet-derived small-EVs, providing a platelet activation assessment independent of the ascites volume. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT). Overall, we presented a high-throughput method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status.
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Project description:Platelet glycoprotein 4 (CD36) (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3]) is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs) and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53-100% identity as compared with 29-32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 'short loops'; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA) contributing to a high gene expression level (6.6 times average). Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate SCARB1 and SCARB2 genes. These suggested that CD36 originated in an ancestral genome and was subsequently duplicated to form three vertebrate CD36 gene family members, SCARB1, SCARB2 and CD36.
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