A novel homozygous mutation (1619delC) in GPIIb gene associated with Glanzmann thrombasthenia, the decay of GPIIb-mRNA and the synthesis of a truncated GPIIb unable to form complex with GPIIIa

2005 ◽  
Vol 93 (05) ◽  
pp. 904-909 ◽  
Author(s):  
Gergely Losonczy ◽  
Nurit Rosenberg ◽  
Csongor Kiss ◽  
János Kappelmayer ◽  
György Vereb ◽  
...  
Keyword(s):  
Stop Codon ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Homozygous Deletion ◽  
Homozygous Mutation ◽  
Truncated Form ◽  
Platelet Surface ◽  
Form Complex ◽  
Glanzmann Thrombasthenia ◽  
Fibrinogen Receptor

SummaryThe absence of agonist-induced platelet aggregation and the lack of fibrinogen receptor (GPIIb/IIIa) on the platelet surface demonstrated that the severe hemorrhagic complications of a child of Romany descent were caused by Glanzmann thrombasthenia. DNA sequencing revealed a novel homozygous deletion of a cytosine (1619delC) in the GPIIb gene causing a frameshift and predicting a novel stop codon at position 533 following 24 altered amino acids. Both parents possessed the same deletion in heterozygous form. The amount of GPIIb mRNA in the patient’s platelets was 0.06% of the amount measured in control platelets. Neither GPIIb nor its truncated form could be detected in the platelets of the patient by Western blotting, while a small amount of GPIIIa was demonstrated. Quantitative flow cytometric analysis showed an elevated number of vitronectin receptors, a component of which is GPIIIa, on the patient’s platelets. The surface expression of vitronectin receptor on thrombasthenic, but not on normal platelets was further increased by activation with thrombin receptor agonist peptide. BHK cells transfected with wild type GPIIIa and mutated GPIIb failed to express any mature GPIIb or pro-GPIIb. Immunoprecipitation with a polyclonal antibody recognizing both GPIIb and GPIIIa recovered a 60 kDa truncated form of GPIIb. This band was absent when immunoprecipitation was carried out with an antibody recognizing GPIIIa, suggesting that the truncated protein, lacking calf-1, calf-2 domains and major part of the thigh domain, is unable to form complex with GPIIIa.

Disulfide bond disruption by a β3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active αIIbβ3

2007 ◽  
Vol 98 (12) ◽  
pp. 1257-1265 ◽  
Author(s):  
Hava Peretz ◽  
Meytal Landau ◽  
Barry Coller ◽  
Abdalla Awidi ◽  
Ronit Mor-Cohen ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Surface Expression ◽  
The Novel ◽  
Platelet Surface ◽  
Glanzmann Thrombasthenia ◽  
Normal Ratio ◽  
Common Founder ◽  
Epidermal Growth ◽  
Reduced Surface ◽  
Constitutively Active

SummaryαIIbβ3 integrin mediates platelet aggregation following its activation. Its absence or dysfunction causes Glanzmann thrombasthenia (GT), an inherited bleeding disorder that is rare worldwide but relatively frequent in several populations with high rates of consanguinity, including Arabs in Israel and Jordan. Cysteine residues in the β3 epidermal growth factor (EGF) domains are involved in αIIbβ3 formation and activation. In this study we present a novel Cys549Arg mutation in β3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of β3 predicting disruption of a conserved disulfide bond between Cys549 and Cys558. Haplotype analysis disclosed a common founder whose age estimate was 120–150 years. Flow cytometry revealed 1–14% of normal αIIbβ3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a β3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant β3 along with normal αIIb demonstrated reduced surface expression of αIIbβ3 by both mutants. The mutants were constitutively active as demonstrated by 20-fold increased binding of the ligand-mimetic antibody PAC-1. Immunoblotting and immunoprecipitation experiments showed reduced β3 and αIIbβ3 expression and a higher than normal ratio of pro-αIIb to mature αIIb. Immunofluorescence experiments showed that β3 and αIIbβ3 were mostly retained in the endoplasmic reticulum. In conclusion, the novel ancestral mutation found in a cluster of Jordanian GT patients disrupts a conserved Cys549-Cys558 bond which results in reduced production of constitutively active αIIbβ3.

Platelet Functions and Risk Prognosis in Myelodysplastic Syndromes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3806-3806
Author(s):  
Nora V. Butta ◽  
Mónica Martín Salces ◽  
Raquel de Paz ◽  
Elena G. Arias Salgado ◽  
Ihosvany Fernández Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndromes ◽  
Surface Expression ◽  
Low Risk ◽  
Control Group ◽  
Platelet Surface ◽  
High Risk Patients ◽  
Fibrinogen Receptor ◽  
Risk Patients

Abstract Abstract 3806 The myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation. The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high). Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count. The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS. Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds. Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions. I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test. CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p<0.05). Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p<0.05); 4161±591 in I-1-risk MDS (p<0.01 versus control and p<0.05 versus low-risk) and 492±184 in high-risk MDS (p<0.01 versus control and p<0.05 versus low-risk). The platelet surface expression of P-selectin induced by 100 μM TRAP was also reduced: 5102±340 AU in controls, 3318±400 AU in low-risk MDS (p<0.05); 1880 ±197 AU in I-1-risk MDS (p<0.05 versus control and versus low-risk), and 1211±130 AU in high-risk MDS (p<0.05 versus control and versus low-risk). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients. Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p<0.05); 575±52 in I-1-risk MDS (p<0.05 versus control and versus low-risk); 611±17 in high-risk MDS (p<0.05 versus control and versus low-risk). Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures: No relevant conflicts of interest to declare.

A naturally occurring mutation near the amino terminus of αIIb defines a new region involved in ligand binding to αIIbβ3

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 180-188 ◽  
Author(s):  
Ramesh B. Basani ◽  
Deborah L. French ◽  
Gaston Vilaire ◽  
Deborah L. Brown ◽  
Fangping Chen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluorescein Isothiocyanate ◽  
Surface Expression ◽  
Chinese Patient ◽  
Platelet Surface ◽  
Glanzmann Thrombasthenia ◽  
Ovary Cells ◽  
Similar Phenotype

Abstract Decreased expression of functional IIbβ3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of IIbP145 in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P145A substitution, whereas a Chinese patient was doubly heterozygous for a P145L substitution. The mutations affect expression levels of surface IIbβ3 receptors on their platelets, which was confirmed by co-transfection of IIbP145A and β3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of IIbβ3 on affected platelets to interact with ligands. Moreover, when IIbP145A and β3 were stably coexpressed in Chinese hamster ovary cells, IIbβ3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate–fibrinogen after IIbβ3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to IIb, induced PF4 secretion, indicating that the mutant IIbβ3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving IIbP145 impair surface expression of IIbβ3 and that the IIbP145A mutation abrogates ligand binding to the activated integrin. A comparative analysis of other IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the IIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

Platelet Apoptosis and Agonist Mediated Activation In Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1866-1866
Author(s):  
Nora V. Butta ◽  
Raquel de Paz ◽  
Mónica Martín Salces ◽  
Ihosvany Fernández Bello ◽  
Elena G. Arias Salgado ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Myelodysplastic Syndromes ◽  
Surface Expression ◽  
Refractory Anemia ◽  
Control Group ◽  
Western Blots ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Bax Protein

Abstract Abstract 1866 Myelodysplastic syndromes (MDS) comprise distinct disorders characterized by dysplastic and ineffective hematopoiesis that seems to be related to an increased apoptosis of bone marrow cells (Nimer, Blood 111: 4841, 2008). Clinical manifestations in MDS range from a diverse degree of anemias, leuko- or thrombocytopenias to severe transfusion-dependent peripheral pancytopenias. Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS. Many of the features of apoptosis such as membrane fragmentation, microvesiculation and phosphatidylserine (PS) exposure are observed during platelet activation to a procoagulant state, raising the possibility that apoptosis may regulate platelet function. The aim of this work was to determine whether a correlation exists between apoptosis and activation processes in platelets from MDS patients. Twenty six patients diagnosed MDS and classified according to WHO-2008 were included: 6 with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), 6 with refractory RA with excess blasts-1 (RAEB-1) and 7 with cytopenia with multilineage dysplasia (RCMD) associated with isolated 5q deletion. Twenty six healthy donors were included as control group. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane PS exposed under basal conditions and after stimulation with a PAR-1 receptor agonist (TRAP, SFLLRN, thrombin receptor-activating peptide 6). Levels of pro- apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis of western blots performed with platelet lysates. Platelet activation was determined through FITC-fibrinogen, FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 mM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (aIIb and b3 subunits) was determined by flow cytometry with specific mAbs. Platelets from RA, RARS and RCMD patients expressed more PS than control ones under basal conditions (p<0.05) as well as after 100 mM TRAP stimulation (p<0.05). Moreover, platelets from these MDS patients expressed more Bax protein than control group (p<0.05). On the other hand, PS exposure and Bax content in platelets from RAEB-1 patients were similar to controls, but they expressed a higher amount of Bcl-2 (p<0.05). No correlation was observed between PS exposure or Bax expression and platelet number. Platelets from all MDS patients showed an impaired activation by TRAP, even when PS exposure was higher than in control group. This diminished response to TRAP was not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients. Our results suggest that platelets from RA, RARS and RCMD patients are more apoptotic than control ones and that a correlation between platelet surface PS and activation does not seem to exist. Moreover, dissimilarity in expression pattern of apoptotic proteins among MDS types indicates differences in the intracellular mechanisms underlying these pathologies. Disclosures: No relevant conflicts of interest to declare.

A Leu262Pro mutation in the integrin β3 subunit results in an αIIb-β3 complex that binds fibrin but not fibrinogen

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Christopher M. Ward ◽  
Anita S. Kestin ◽  
Peter J. Newman
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Surface Expression ◽  
Fibrin Clot ◽  
Wild Type ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
293 Cells ◽  
Platelet Disorder

Abstract Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Proβ3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type β3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for IIbβ3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding β3 Leu262 may maintain β3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.

Two Different β3 Cysteine Substitutions Alter αIIbβ3 Maturation and Result in Glanzmann Thrombasthenia

2002 ◽  
Vol 88 (07) ◽  
pp. 104-110 ◽  
Author(s):  
S. Milet-Marsal ◽  
C. Breillat ◽  
O. Peyruchaud ◽  
P. Nurden ◽  
R. Combrié ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Transient Expression ◽  
Double Mutant ◽  
Direct Sequencing ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Homozygous Mutation ◽  
Glanzmann Thrombasthenia ◽  
Exon 10

SummaryWe report the defects responsible for Glanzmann thrombasthenia in two patients showing traces of abnormally migrating platelet β3 in immunoblotting. Using PCR-SSCP and direct sequencing, we identified a novel homozygous mutation in exon 10 of the β3 gene of patient 1 which gave a C457 to Y amino acid substitution. A C542 to R substitution in β3 of patient 2 was previously reported by us. These cysteines are present in EGF-domains 1 and 3 respectively of β3. We therefore constructed mutants carrying substitutions on cysteine residues in each of the first three EGF domains of β3, C457, C495 and C542 respectively. Transient expression of these mutants in COS-7 cells, including the C542 and C547 double mutant, proved that disulfide disruption directly affects cell surface expression of the integrin. We then showed by metabolic (35S) labeling and Endo-H glycosidase treatment that these substitutions strongly affected complex maturation within the cell.

A Leu262Pro mutation in the integrin β3 subunit results in an αIIb-β3 complex that binds fibrin but not fibrinogen

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Christopher M. Ward ◽  
Anita S. Kestin ◽  
Peter J. Newman
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Surface Expression ◽  
Fibrin Clot ◽  
Wild Type ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
293 Cells ◽  
Platelet Disorder

Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Proβ3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type β3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for IIbβ3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding β3 Leu262 may maintain β3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.

Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 770-779 ◽  
Author(s):  
AD Michelson ◽  
PA Ellis ◽  
MR Barnard ◽  
GB Matic ◽  
AF Viles ◽  
...  
Keyword(s):  
Whole Blood ◽  
Actin Polymerization ◽  
Flow Cytometric Analysis ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Surface Glycoprotein ◽  
Platelet Surface ◽  
Granule Secretion

Abstract In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

scholarly journals Case Report: A Chinese Family of Woodhouse-Sakati Syndrome With Diabetes Mellitus, With a Novel Biallelic Deletion Mutation of the DCAF17 Gene

2021 ◽  
Vol 12 ◽  
Author(s):  
Min Zhou ◽  
Ningjie Shi ◽  
Juan Zheng ◽  
Yang Chen ◽  
Siqi Wang ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Stop Codon ◽  
Signs And Symptoms ◽  
Homozygous Deletion ◽  
Chinese Family ◽  
Homozygous Mutation ◽  
Chinese Han ◽  
C Peptide ◽  
Whole Exome ◽  
Peptide Release

Woodhouse–Sakati syndrome (WSS) (OMIM#241080) is a rare multi-system autosomal recessive disease with homozygous mutation of the DCAF17 gene. The main features of WSS include diabetes, hypogonadism, alopecia, deafness, intellectual disability and progressive extrapyramidal syndrome. We identified a WSS family with a novel DCAF17 gene mutation type in China. Two unconsanguineous siblings from the Chinese Han family exhibiting signs and symptoms of Woodhouse-Sakati syndrome were presented for evaluation. Whole-exome sequencing revealed a homozygous deletion NM_025000.4:c.1488_1489delAG in the DCAF17 gene, which resulted in a frameshift mutation that led to stop codon formation. We found that the two patients exhibited low insulin and C-peptide release after glucose stimulation by insulin and C-peptide release tests. These findings indicate that the DCAF17 gene mutation may cause pancreatic β cell functional impairment and contribute to the development of diabetes.

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