A novel 196 Leu to Pro substitution in the β3 subunit of the αIIbβ3 integrin in a patient with a variant form of Glanzmann thrombasthenia

Nurden, Alan T.; Ruan, Jian; Pasquet, Jean‐Max; Gauthier, Bruno; Combrié, Robert; Kunicki, Thomas J.; Nurden, Paquita

doi:10.1080/09537100220122466

Cited by 20 publications

(34 citation statements)

References 29 publications

(31 reference statements)

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“…Procedures were as previously described by us. 26,27 Activation of ␣IIb␤3 was studied on platelets in whole blood incubated with 10 M ADP and evaluated using PAC-1 (Becton Dickinson, Pont de Claix, France). Expression of P-selectin and granulophysin at the platelet surface was studied before and after activation of platelets by 50 M TRAP using, respectively, the MoAbs VH10 prepared by the Bordeaux Laboratory and MoF11 kindly given by Dr P. Vincendeau (Bordeaux University, France).…”

Section: Flow Cytometrymentioning

confidence: 99%

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Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

Nurden

Jandrot‐Perrus

Combrié

et al. 2004

Blood

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We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking ␣-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor ␥-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C␥2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the

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Section: Flow Cytometrymentioning

confidence: 99%

“…FITC-labeled F(abЈ) 2 fragments of a sheep antibody to mouse IgG (Silenus) were employed to detect murine MoAbs. 26,27 Platelets were analyzed in a FACScan (Becton Dickinson). Gating to select most platelets was based on preliminary determinations of forward and wide-angle light scatter.…”

Section: Flow Cytometrymentioning

confidence: 99%

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

Nurden

Jandrot‐Perrus

Combrié

et al. 2004

Blood

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“…In ␤3 integrins, mutation of Leu-196 to proline differentially affected ␣IIb␤3 and ␣V␤3 integrin expression and ability to interact with immobilized ligands. ␣V␤3 integrinmediated spreading on fibrinogen was blocked by this mutation, whereas ␣IIb␤3 activation by dithiothreitol, antibody, or platelet activation was blocked (41,42). Given that mutation of this residue in ␤PS to phenylalanine has the opposite effect of mutation in ␣IIb␤3 to proline, one of these substitutions probably does more than simply remove the well conserved leucine at this position, so its contribution to integrin function is still in question and will require further analysis.…”

Section: ␣Ps2␤ps Integrin Ligand Affinitymentioning

confidence: 99%

Identification of Integrin β Subunit Mutations That Alter Affinity for Extracellular Matrix Ligand

Kendall¹,

Mukai²,

Jannuzi³

et al. 2011

Journal of Biological Chemistry

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We examined over 50 mutations in the Drosophila ␤PS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the ␤ tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of ␤PS and ␣PS2C reveal different effects on ligand binding from those observed for ␣IIb␤3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the ␤PS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.Integrin cell surface receptors are important for morphogenetic events in normal development and are also critical for adult and many disease-related processes as well (1). The integrin ␣␤ heterodimer is a dynamic receptor, whose activity is regulated by interactions with extracellular, intracellular, and other transmembrane proteins. This regulation can change the affinity of individual integrin molecules or alter the clustering of integrins such that their avidity is altered. Experiments utilizing conformation-specific antibodies, high resolution x-ray, NMR, small angle neutron scattering, molecular dynamics, steered molecular dynamics, and electron microscopy are elucidating how the ␣ and ␤ subunits are organized and change in response to activating conditions and ligand binding. One goal of the current work on integrin biology is to understand how the structural information gained on isolated integrins relates to integrin function in a cellular context. Therefore, models for integrin activation are being tested by site-directed mutagenesis (reviewed in Refs. 2-4).We have used a complementary approach of first identifying amino acids in the Drosophila integrin ␤PS subunit that alter its function in the whole organism and then examining the effects of these mutations at the cellular and molecular level. Previous work demonstrated that myospheroid (mys, encoding ␤PS integrin) hypomorphic mutations displayed stronger phenotypes at higher temperatures (5). Therefore, we screened for mutations in mys that produce viable flies at low temperatures but lethality at high temperatures. Because null alleles of mys are complet...

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“…A sister died from nasal bleeding when she was a child. Whereas each of the three family members had somewhat decreased but 450% expression of IIbb3 in their platelets, pat 1 only possessed of the order of 8% of the normal platelet content of IIbb3 [1]. Nevertheless, unlike for classic type I GT, his platelets contained detectable amounts of stored and presumably endocytosed Fg.…”

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confidence: 93%

“…(Received 11 March 2010; accepted 11 March 2010) To the Editor In 1999 we published the case of a male patient from the Bordeaux area of France with variant-type Glanzmann thrombasthenia (GT) linked to a heterozygous c.685T4C transition in exon 5 of ITGB3 giving rise to a L196P (L222P, including the leader sequence) substitution [1,2]. This mutation was detected following an initial screening of the exons and splice sites of the ITGB3 and ITGA2B genes of the propositus ( pat 1 here) by single-strand conformation polymorphism analysis of PCR products.…”

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confidence: 99%