scholarly journals Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 881-888 ◽  
Author(s):  
PF Bray ◽  
MA Shuman
Keyword(s):  
Genetic Defect ◽  
Large Family ◽  
Northern Analysis ◽  
Specific Gene ◽  
Carrier Status ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Glanzmann's Thrombasthenia ◽  
Ecori Restriction ◽  
Adhesive Interactions

Abstract The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3′ GPIIIa cDNA. However, in one family, a 5′ 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5′ 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.

scholarly journals Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 881-888 ◽  
Author(s):  
PF Bray ◽  
MA Shuman
Keyword(s):  
Genetic Defect ◽  
Large Family ◽  
Northern Analysis ◽  
Specific Gene ◽  
Carrier Status ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Glanzmann's Thrombasthenia ◽  
Ecori Restriction ◽  
Adhesive Interactions

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3′ GPIIIa cDNA. However, in one family, a 5′ 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5′ 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.

Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

1995 ◽  
Vol 73 (05) ◽  
pp. 756-762 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
Masamichi Shiraga ◽  
Yoshio Kanayama ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Nucleotide Sequence Analysis ◽  
Type I ◽  
Normal Amount ◽  
Immunoblot Assay ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

Application of GPIIIa Gene Taq I Polymorphism to Determination of Carrier Status in Glanzmann’s Thrombasthenia Families of Chinese Origin

1993 ◽  
Vol 69 (01) ◽  
pp. 064-069 ◽  
Author(s):  
Changgeng Ruan ◽  
Jianming Gu ◽  
Xiaodong Wang ◽  
Xiaohong Chu ◽  
Junliang Pan
Keyword(s):  
Carrier Status ◽  
Glanzmann’S Thrombasthenia ◽  
Chinese Origin ◽  
Length Polymorphisms ◽  
Glanzmann's Thrombasthenia ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Asymptomatic Subjects ◽  
Autosomal Recessive Manner

SummaryGlanzmann’s thrombasthenia is a bleeding disorder caused by qualitative and/or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. The disease is inherited in an autosomal recessive manner. In this paper, cDNA probes were used to study restriction fragment length polymorphisms (RFLPs) in GPIIIa gene. A Taq I polymorphism was identified and this RFLP was composed of variant bands of 6.5 Kb/4.0 and 2.5 Kb with a frequency of 0.46/0.54 in Chinese population. The Taq I polymorphism was further localized by polymerase chain reaction (PCR) method to exon VIII of the GPIIIa gene. In two Glanzmann’s thrombasthenia families, the Taq I RFLP studied by both Southern blotting and PCR methods identified the defective GPIIIa gene inherited by patients, and determined the genotype of asymptomatic subjects. Analysis of this Taq I polymorphism by PCR method should be potentially useful in future for the carrier detection and prenatal diagnosis in Glanzmann’s thrombasthenia families.

Case 6: A 2-year-old boy with mucocutaneous bleeding – Glanzmann’s Thrombasthenia. The role of the fibrinogen-receptor glycoprotein IIb–IIIa in platelet function

1999 ◽  
Vol 56 (9) ◽  
pp. 495-498
Author(s):  
Alberio ◽  
Hirt
Keyword(s):  
Platelet Function ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Glanzmann's Thrombasthenia ◽  
Diagnostik Und Therapie ◽  
Mucocutaneous Bleeding

Der Fall eines Patienten mit einer seit Geburt vorhandenen mukokutanen Blutungsneigung wird vorgestellt. Es wurde eine Thrombasthenie Glanzmann diagnostiziert. An diesem Beispiel werden wichtige Aspekte der Diagnostik und Therapie von Thrombozytenfunktionsstörungen beschrieben. Zudem wer-den die Thrombozytenbeteiligung im Blutstillungsvorgang sowie die Rolle des thrombozytären Fibrinogen-Rezeptors Glykoprotein IIb–IIIa im Rahmen der primären Hämostase dargestellt.

scholarly journals Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1108-1115 ◽  
Author(s):  
WC Kouns ◽  
B Steiner ◽  
TJ Kunicki ◽  
S Moog ◽  
J Jutzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Synthetic Peptides ◽  
Tertiary Structure ◽  
Divalent Cations ◽  
Ligand Binding Site ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Glanzmann's Thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211–221. However, we recently showed that synthetic peptides corresponding to amino acids 211–221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214–221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43–5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.

scholarly journals Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1108-1115 ◽  
Author(s):  
WC Kouns ◽  
B Steiner ◽  
TJ Kunicki ◽  
S Moog ◽  
J Jutzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Synthetic Peptides ◽  
Tertiary Structure ◽  
Divalent Cations ◽  
Ligand Binding Site ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Glanzmann's Thrombasthenia

Abstract One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211–221. However, we recently showed that synthetic peptides corresponding to amino acids 211–221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214–221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43–5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

Stimulated Glanzmann’s Thrombasthenia Platelets Produce Microvesicles

1995 ◽  
Vol 74 (06) ◽  
pp. 1533-1540 ◽  
Author(s):  
Pål André Holme ◽  
Nils Olav Solum ◽  
Frank Brosstad ◽  
Nils Egberg ◽  
Tomas L Lindahl
Keyword(s):  
Complement System ◽  
Shape Change ◽  
Annexin V ◽  
Platelet Surface ◽  
Glanzmann’S Thrombasthenia ◽  
Large Numbers ◽  
Glanzmann's Thrombasthenia ◽  
Series Of Experiments ◽  
The Complement System ◽  
Number Of Particles

SummaryThe mechanism of formation of platelet-derived microvesicles remains controversial.The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann’s thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 μM), and the thrombin receptor agonist peptide SFLLRN (100 μM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 μM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 ±2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 ± 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 μM SFLLRN gave 16.3 ± 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 ± 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.

Diversity of Glycoprotein Deficiencies in Glanzmann’s Thrombasthenia

1985 ◽  
Vol 54 (03) ◽  
pp. 626-629 ◽  
Author(s):  
M Meyer ◽  
F H Herrmann
Keyword(s):  
High Resolution ◽  
Gel Electrophoresis ◽  
Type Ii ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Structural Variant ◽  
Glanzmann’S Thrombasthenia ◽  
Crossed Immunoelectrophoresis ◽  
Glanzmann's Thrombasthenia ◽  
Platelet Proteins

SummaryThe platelet proteins of 9 thrombasthenic patients from 7 families were analysed by high resolution two-dimensional gel electrophoresis (HR-2DE) and crossed immunoelectrophoresis (CIE). In 7 patients both glycoproteins (GPs) IIb and Ilia were absent or reduced to roughly the same extent. In two related patients only a trace of GP Ilb-IIIa complex was detected in CIE, but HR-2DE revealed a glycopeptide in the position of GP Ilia in an amount comparable to type II thrombasthenia. This GP Ilia-like component was neither recognized normally by anti-GP Ilb-IIIa antibodies nor labeled by surface iodination. In unreduced-reduced two-dimensional gel electrophoresis two components were observed in the region of GP Ilia. The assumption of a structural variant of GP Ilia in the two related patients is discussed.

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