scholarly journals Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2339-2347 ◽  
Author(s):  
SD Wright ◽  
K Michaelides ◽  
DJ Johnson ◽  
NC West ◽  
EG Tuddenham
Keyword(s):  
Normal Subjects ◽  
Immunoblot Analysis ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Missense Mutations ◽  
Coding Region ◽  
Giant Platelets ◽  
Double Heterozygosity ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

Abstract Bernard-Soulier syndrome (BSS) giant platelets have defective and/or deficient glycoprotein (GP) Ib/IX complexes, causing absent ristocetin- induced aggregation, defective interaction with von Willebrand factor, morphologic abnormality, and a clinical bleeding tendency. Recently several mutations have been described in the platelet GPIb alpha gene in individuals exhibiting the BSS phenotype. We have studied a family with classical BSS, and have excluded lesions at the GPIb alpha locus by restriction fragment length polymorphism linkage analysis. Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine. Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif. Both mutations create new recognition sites for the enzyme Fnu 4H1; therefore, this enzyme was used to screen 60 normal subjects (120 alleles). Neither mutation was detected in any subject other than direct relatives of the affected individuals. Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity. We propose that expression of abnormal GPIX prevents stable assembly of the GPIb/IX complex, causing BSS in the doubly heterozygous individuals in this family.

scholarly journals Double heterozygosity for mutations in the platelet glycoprotein IX gene in three siblings with Bernard-Soulier syndrome

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2339-2347 ◽  
Author(s):  
SD Wright ◽  
K Michaelides ◽  
DJ Johnson ◽  
NC West ◽  
EG Tuddenham
Keyword(s):  
Normal Subjects ◽  
Immunoblot Analysis ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Missense Mutations ◽  
Coding Region ◽  
Giant Platelets ◽  
Double Heterozygosity ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

Bernard-Soulier syndrome (BSS) giant platelets have defective and/or deficient glycoprotein (GP) Ib/IX complexes, causing absent ristocetin- induced aggregation, defective interaction with von Willebrand factor, morphologic abnormality, and a clinical bleeding tendency. Recently several mutations have been described in the platelet GPIb alpha gene in individuals exhibiting the BSS phenotype. We have studied a family with classical BSS, and have excluded lesions at the GPIb alpha locus by restriction fragment length polymorphism linkage analysis. Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine. Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif. Both mutations create new recognition sites for the enzyme Fnu 4H1; therefore, this enzyme was used to screen 60 normal subjects (120 alleles). Neither mutation was detected in any subject other than direct relatives of the affected individuals. Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity. We propose that expression of abnormal GPIX prevents stable assembly of the GPIb/IX complex, causing BSS in the doubly heterozygous individuals in this family.

BERNARD-SOULIER SYNDROME: WHOLE BLOOD DIAGNOSTIC ASSAYS OF PLATELETS

1987 ◽  
Author(s):  
W L Nichols ◽  
S E Kaese ◽  
D A Gastineau ◽  
L A Otteman ◽  
E J W Bowie
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Platelet ◽  
Laboratory Diagnosis ◽  
Light Transmittance ◽  
Immunocytochemical Staining ◽  
Platelet Glycoprotein ◽  
Giant Platelets ◽  
Bernard Soulier Syndrome

Diagnosis of Bernard-Soulier syndrome (BSS) is complicated by the difficulty of separating the giant platelets from other blood cells to pursue analyses of platelet function and structure. We report on the utility of three whole blood assay techniques for diagnosis of a patient with BSS. To our knowledge, these three techniques have not been simultaneously applied or compared for efficacy in laboratory diagnosis of BSS. (1) Whole blood platelet aggregation responses, studied with an electrical impedence aggregometer, were equivalent to those more laboriously obtained using platelet-rich plasma prepared by unit gravity sedimentation, studied with an optical light transmittance aggregometer. Platelet aggregation responses were normal with ADP or collagen stimulation, and absent with Ristocetin or bovine plasma stimulation. (2) Whole blood radioimmunoassay of platelet glycoprotein (GP) expression was performed using iodinated murine monoclonal antibodies HP1-1D (anti-GP IIb/IIIa) and 6D1 (anti-GPlb, kindly supplied by Dr. Barry Coller, Stony Brook, NY). After incubation with citrated whole blood, centrifugation was used to separate cell-bound antibody which was quantitated with a gamma counter. The patient’s whole blood had a normal level of cell-bound GP Ilb/IIIa, but a markedly reduced level of cell-bound GP lb (5% of normal mean; n = 20). (3) Whole blood smear immunocytochemical staining with the monoclonals (indirect immuno-alkaline phosphatase technique), and qualitative analysis by light microscopy, revealed a marked reduction of GP lb expression by the patient’s giant platelets, whereas GP Ilb/IIIa expression was normal. This latter technique might be especially valuable as a screening technique when the patient is not directly available for laboratory study. Together with the patient’s life-long history of thrombocytopenia and moderate bleeding diathesis, and other laboratory observations including markedly prolonged bleeding times and reduced whole blood prothrombin consumption, these data established diagnosis of BSS. We conclude that these three relatively simple assays of platelets in whole blood should be of particular value in the laboratory differential diagnosis of patients with congenital thrombocytopenias and giant platelet syndromes.

scholarly journals Pseudo-Bernard-Soulier syndrome: thrombocytopenia caused by autoantibody to platelet glycoprotein Ib

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 428-431
Author(s):  
DV Devine ◽  
MS Currie ◽  
WF Rosse ◽  
CS Greenberg
Keyword(s):  
Immunoglobulin G ◽  
Adenosine Diphosphate ◽  
Platelet Glycoprotein ◽  
Laboratory Studies ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Immune Mediated ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

The Bernard-Soulier syndrome is an inherited bleeding disorder that is due to a deficiency in platelet glycoprotein Ib. Bernard-Soulier platelets fail to agglutinate in response to ristocetin despite normal levels of factor VIII:von Willebrand factor. We report a patient who developed severe refractory thrombocytopenia postsurgically while receiving procainamide therapy. Thrombocytopenia was immune mediated since the patient's platelets bore high levels of antiplatelet antibody. Radioimmunoprecipitation studies demonstrated that the autoantibodies had specificity for platelet glycoproteins Ib and V as well as platelet HLA. The patient's plasma as well as purified immunoglobulin G completely inhibited the ristocetin-induced aggregation of normal platelets but did not inhibit adenosine diphosphate-induced aggregation. The laboratory studies revealed that this patient suffered from antibody-mediated thrombocytopenia with unusual characteristics that we have called pseudo-Bernard-Soulier syndrome.

Compound Heterozygosity (554-589 del, C515-T Transition) in the Platelet Glycoprotein Ibα Gene in a Patient with a Severe Bleeding Tendency

1999 ◽  
Vol 81 (04) ◽  
pp. 486-492 ◽  
Author(s):  
Maurizio Margaglione ◽  
Giovanna D’Andrea ◽  
Elvira Grandone ◽  
Vincenzo Brancaccio ◽  
Aldo Amoriello ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Normal Function ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Bleeding Tendency ◽  
Giant Platelets ◽  
Novel Variant ◽  
Leucine Rich Repeats ◽  
Von Willebrand

SummaryGiant platelets in the blood smear, absent in vitro platelet agglutination in response to ristocetin, and normal aggregation, ATP secretion and thromboxane B2 formation were found in a young patient with a life-long bleeding tendency. Ristocetin-induced von Willebrand factor binding to her platelets was less than 10% of normal. Flow cytometric analysis with monoclonal antibodies LJ-Ib-1, LJ-Ib-10, and LJ-P3 was consistent with the latter finding. SDS-PAGE analysis of solubilized platelets showed a marked reduction of the platelet glycoprotein (GP) Ibα. Genetic characterisation demonstrated that the patient and her father were heterozygous for a deletion of 36 nucleotides (positions 554-589) leading to a mutant GPIbμ (deletion of aminoacids from residue 169 to 180 and a Glu → Lys substitution at residue 181). In addition, a C → T transition at nucleotide 515 in the other allele of the GPIbα gene was found in the patient and in her mother that results in the substitution of alanine for valine in codon 156 (Bernard-Soulier type Bolzano). These variations occurred within the VI and VII leucine-rich repeats. The novel variant of Bernard-Soulier syndrome identified further suggests that the integrity of leucine-rich repeats is important for normal function of the GP Ib-IX-V receptor complex.

scholarly journals Pseudo-Bernard-Soulier syndrome: thrombocytopenia caused by autoantibody to platelet glycoprotein Ib

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 428-431 ◽  
Author(s):  
DV Devine ◽  
MS Currie ◽  
WF Rosse ◽  
CS Greenberg
Keyword(s):  
Immunoglobulin G ◽  
Adenosine Diphosphate ◽  
Platelet Glycoprotein ◽  
Laboratory Studies ◽  
Antiplatelet Antibody ◽  
Glycoprotein Ib ◽  
Immune Mediated ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

Abstract The Bernard-Soulier syndrome is an inherited bleeding disorder that is due to a deficiency in platelet glycoprotein Ib. Bernard-Soulier platelets fail to agglutinate in response to ristocetin despite normal levels of factor VIII:von Willebrand factor. We report a patient who developed severe refractory thrombocytopenia postsurgically while receiving procainamide therapy. Thrombocytopenia was immune mediated since the patient's platelets bore high levels of antiplatelet antibody. Radioimmunoprecipitation studies demonstrated that the autoantibodies had specificity for platelet glycoproteins Ib and V as well as platelet HLA. The patient's plasma as well as purified immunoglobulin G completely inhibited the ristocetin-induced aggregation of normal platelets but did not inhibit adenosine diphosphate-induced aggregation. The laboratory studies revealed that this patient suffered from antibody-mediated thrombocytopenia with unusual characteristics that we have called pseudo-Bernard-Soulier syndrome.

The genetic defect in two well-studied cases of Bernard-Soulier syndrome: a point mutation in the fifth leucine-rich repeat of platelet glycoprotein Ib alpha

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3805-3814 ◽  
Author(s):  
C Li ◽  
SE Martin ◽  
GJ Roth
Keyword(s):  
Point Mutation ◽  
Genetic Defect ◽  
Pcr Amplification ◽  
Mutant Gene ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Molecular Defect ◽  
Wild Type ◽  
Giant Platelets ◽  
Bernard Soulier Syndrome

Bernard-Soulier syndrome (B-Ss) is a rare congenital bleeding disorder caused by abnormal giant platelets, thrombocytopenia, and defective glycoprotein (GP) Ib-V-IX, the adhesion receptor for von Willebrand factor (vWF). This report describes the molecular defect in two related individuals with well-established B-Ss whose platelets exhibit decreased GPIb-IX and normal GPV on their surfaces. The GPIb-V-IX genes of the two patients were analyzed by Southern blotting, hetero-duplex analysis, and polymerase chain reaction (PCR) amplification/sequencing. A point mutation was found in codon 129 of the GPIb alpha gene that results in the substitution of proline for leucine in the first position of the fifth leucine-rich glycoprotein repeat of the mature gene product. The mutation (CTC: leucine, wild-type to CCC: proline, mutant) eliminates a Sac I restriction site, facilitating analysis of the mutation in the propositi (both homozygotes), unaffected family members (8 heterozygotes and 8 wild-type), and 58 normal controls (116 wild-type alleles). The status of the genomes was confirmed by the sequencing of platelet cDNA. The mutation does not affect transcription of the Ib-IX genes, as estimated by PCR and Northern blot analysis, but it does inhibit surface expression of the receptor as assessed by transient transfection of mutant and wild-type GPIb alpha genes into mouse Ib beta-IX L cells. Many of the cells (43%) transfected with the normal gene express surface GPIb alpha, whereas untransfected cells and those transfected with the mutant gene lack surface GPIb alpha entirely. Patient platelets were tested both for vWF binding in the presence of ristocetin and for surface GPIb-IX expression. In these instances, despite their inability to agglutinate with ristocetin and vWF, patient platelets exhibit about 40% of normal vWF binding and 40% of normal Ib-IX surface antigens. The results suggest that the described mutation (GPIb alpha: Leu129 -> Pro) affects the conformation of the GPIb-V-IX receptor, alters its availability on platelet surfaces, and causes the observed Bernard-Soulier phenotype.

scholarly journals A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 570-577 ◽  
Author(s):  
CG Ruan ◽  
XP Du ◽  
XD Xi ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Scatchard Analysis ◽  
Type I ◽  
Glycoprotein Ib ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

Abstract A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

Bernard–Soulier syndrome with a homozygous 13 base pair deletion in the signal peptide-coding region of the platelet glycoprotein Ibβ gene

2003 ◽  
Vol 14 (4) ◽  
pp. 387-394 ◽  
Author(s):  
Reiko Watanabe ◽  
Toshiyuki Ishibashi ◽  
Yurie Saitoh ◽  
Tsutomu Shichishima ◽  
Yukio Maruyama ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Base Pair ◽  
Platelet Glycoprotein ◽  
Coding Region ◽  
Base Pair Deletion ◽  
Bernard Soulier Syndrome

THROMBASTHENIA WITH AN ABNORMAL PLATELET MEMBRANE GPIIb OF DIFFERENT MOLECULAR WEIGHT

1987 ◽  
Author(s):  
M Moroi ◽  
S M Jung ◽  
N Yoshida ◽  
N Aoki ◽  
K Tanoue
Keyword(s):  
Molecular Weight ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Single Chain ◽  
Fibrinogen Receptor ◽  
Antibody Staining ◽  
Fibrinogen Binding ◽  
Sds Page ◽  
Induced Aggregation ◽  
Different Molecular Weight

We describe several individuals with abnormal platelet glycoprotein lib (GPIIb) of different molecular weight (MW), a novel defect in that previously reported thrombasthenics have not shown GPIIb molecules of abnormal MW. Patient 1 shows 2 bands of GPIIb (one with normal MW and one with abnormal MW) in SDS-PAGE (lectin and antibody staining). This patient has a mild bleeding tendency, and her platelets did not aggregate in response to ADP (2-10 μM) and had severely depressed collagen induced aggregation ,but responded normally to ristocetin. The MW of the abnormal GPIIb band was calculated to be 122 KDa (compared to the 129 kDa of normal GPIIb) in non-reduced SDS-PAGE; the MW was 128 kDa (compared to the normal 118 kDa) in reduced SDS-PAGE. The amount of each GPIIb band, calculated from densitometry, was approximately 35% for the GPIIb of abnormal MW and 20% for the normal MW GPIIb, relative to the amounts of GPIIb found in normal individuals. Assays of fibrinogen binding to the patient`s platelets indicate that they contain 25% of the fibrinogen receptor as compared to normal platelets. Crossed immunoelectrophoresis showed the formation of the GPIIb/IIIa complex, which was found to consist mostly of normal MW GPIIb and GPIIIa. The patient's father showed decreased aggregability in response to ADP, and his platelets also contained abnormal and normal GPIIb. The father's platelets, however, contained about 50% normal GPIIb as compared to normal platelets, and his platelets have about 50% the number of fibrinogen receptors compared to normal platelets. From these results, the patient was suggested to have heterozygous GPIIb molecules, and the observed defects in aggregability would be attributable to the decrease of normal GPIIb to 20-30% of the normal level. Quite recently, we found another thrombasthenic individual who has homozygous abnormal GPIIb molecules. Electrophoretic mobilities of his abnormal GPIIb on SDS-PAGE indicated that the abnormal GPIIb's were very similar between the 2 cases, and two-dimensional, unreduced-reduced SDS-PAGE suggested that the abnormal GPIIb consists of a single chain.

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