A 1063G→A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

Tao, Jianming; Arias-Salgado, Elena G; González-Manchón, Consuelo; Iruı́n, Gema; Butta, Nora; Ayuso, Matilde Sánchez; Parrilla, Roberto L.

doi:10.1111/j.1365-2141.2000.02423.x

Cited by 9 publications

(10 citation statements)

References 32 publications

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“…In support of our findings concerning the importance of region 313-332 in fibrinogen finding, two naturally occurring mutations (E324K and R327H) have been reported in patients with Glanzmann thrombasthenia [46][47][48][49]. Moreover, it has been shown that the peptide LSARLAF [50] binds to complementary region 315-321 of a IIb and induces a IIb b 3 conformational change and platelet aggregation [50,51].…”

Section: Discussionsupporting

confidence: 86%

Mapping the binding domains of the α_IIb subunit

Biris¹,

Abatzis²,

Mitsios³

et al. 2003

European Journal of Biochemistry

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a IIb b 3 , a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligandbinding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the a IIb subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of a IIb potentially involved in the platelet-aggregation event. Regions a IIb 313-332, a IIb 265-284 and a IIb 57-64 of a IIb b 3 were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of a IIb b 3 . Furthermore, a IIb 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in a IIb 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.Keywords: a IIb -binding domains; a IIb mapping; plateletaggregation inhibitors; a IIb b 3 receptor; integrin inhibitors.The integrin family of adhesive protein receptors, composed of noncovalently associated a and b subunits, participates in a number of diverse functions ranging from embryogenesis to cellular aggregation, and differentiation to tumor cell growth and metastasis [1][2][3][4][5]. Integrin receptors consist of at least 20 members composed of different combinations of a and b subunits with distinct ligand-recognition specificity [6].The integrin receptor a IIb b 3 is the most abundant glycoprotein on platelet plasma-membranes. This receptor binds to adhesive proteins, such as fibrinogen, von Willebrand factor, fibronectin, and vitronectin, via the recognition of short amino acid sequences, including the ubiquitous motif RGD, as well as the HHLGGAKQAGDV sequence of the fibrinogen c-chain [7,8]. Binding studies suggest that platelet activation (e.g. by ADP) induces conformational changes of a IIb b 3 , which result in higher affinity to fibrinogen, an event essential for platelet aggregation and thrombus formation [9,10]. mAbs recognizing specific epitopes on the extracellular domains of both subunits are also able to induce/stabilize conformational changes of a IIb b 3 , which increase the affinity of the receptor for its ligands [11][12][13].The discovery that the RGD sequence is present in a surprisingly large number of adhesive proteins, serving div...

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Section: Discussionsupporting

confidence: 86%

Mapping the binding domains of the α_IIb subunit

Biris¹,

Abatzis²,

Mitsios³

et al. 2003

European Journal of Biochemistry

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“…, 1993). Our findings showed mutation in exon 12 of the ITGA2B, it is to be noted that many mutations have been described in the literatures in this exon 12 of ITGA2B (Tao et al. , 2000; Peretz et al.…”

Section: Discussionsupporting

confidence: 70%

Role of RFLP using TspRI for carrier detection in Glanzmann’s thrombasthenia: a report on two families

Kannan

Yadav

Ahmad

et al. 2010

Int J Lab Hematology

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Glanzmann's thrombasthenia (GT) was diagnosed in two patients who presented with bleeding manifestations accompanied by absent platelet aggregation, secondary to adenosine-5'-diphosphate, adrenaline, arachidonic acid and collagen. Flow cytometry analysis for GPIIb/IIIa expression was done using CD61 and CD41 markers in these patients and their family members including siblings. The patients were sub typed as Type I as he had absent glycoproteins (GP) IIb/IIIa. Family studies by flow cytometry showed reduced GPII/IIIa expression in both the parents and one sibling. However, western blot showed abnormal GPIIb protein in all the family members including siblings. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. Patients' DNA was analyzed for mutation in both the GPIIb and GPIIIa genes by conformational sensitive gel electrophoresis (CSGE), followed by sequencing. CSGE showed defect in exon 12 and the promoter region of GPIIb. By sequence, it was confirmed that both the mutations were homozygous one was c.1028T>C and the other one was M33320.1:g.951G>A. For one of these mutations, restriction fragment length polymorphism (RFLP) was developed to look for the same mutation in all the family members. RFLP was developed using a restriction enzyme (TspRI) against the patient's mutation, c.1028T>C in exon 12 of GPIIb gene. RFLP followed by gel electrophoresis revealed that the mutation was heterozygous in all the family members. The findings by RFLP were double confirmed by direct DNA sequencing of the exon 12 in all the family members. Thus, TspRI marker may be used as a RFLP marker to predict the carriers in GT families, if the patients' mutation status is identified.

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“…Genetic studies focused on the GPIIIa as defects in this subunit often give rise to a defective receptor, whereas mutations in GPIIb generally effect receptor expression (Tao et al , 2000). Indeed, two mutations in GPIIIa were found.…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in either the GPIIb or GPIIIa responsible for GT lead to defects in mRNA splicing (Burk et al , 1991; Newman et al , 1991; Iwamoto et al , 1994; Jin et al , 1996), mRNA stability (Kato et al , 1992), divalent cation binding (Poncz et al , 1994; Basani et al , 1996), subunit association (Bajt et al , 1992; Lanza et al , 1992; Tao et al , 2000), intracellular trafficking (Wilcox et al , 1994), ligand binding (Loftus et al , 1990; Ward et al , 2000) and integrin‐mediated signal transduction (Wang et al , 1997). In the Strasbourg variant of GT (GPIIIa Arg214Tyr), the receptor retained the ability to bind RGD ligands but did not recognize fibrinogen (Lanza et al , 1992).…”

Section: Discussionmentioning

confidence: 99%

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

et al. 2001

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We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid‐phase fibrinogen and binding affinity for the RGD‐mimetic 3H‐SC52012, a non‐peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [3H]‐SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3′ end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [3H]‐SC52012 with a higher affinity than wild‐type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.

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A 1063G→A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

Cited by 9 publications

References 32 publications

Mapping the binding domains of the α_IIb subunit

Mapping the binding domains of the α_IIb subunit

Role of RFLP using TspRI for carrier detection in Glanzmann’s thrombasthenia: a report on two families

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

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A 1063G→A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

Cited by 9 publications

References 32 publications

Mapping the binding domains of the αIIb subunit

Mapping the binding domains of the αIIb subunit

Role of RFLP using TspRI for carrier detection in Glanzmann’s thrombasthenia: a report on two families

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

Contact Info

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Mapping the binding domains of the α_IIb subunit

Mapping the binding domains of the α_IIb subunit