The Experts below are selected from a list of 348 Experts worldwide ranked by ideXlab platform
Marco Cattaneo - One of the best experts on this subject based on the ideXlab platform.
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Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients
Scientific Reports, 2016Co-Authors: Christian A. Di Buduo, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Maria Adele Alberelli, Raffaele Landolfi, Marco Cattaneo, Paola R. Lev, Alessandra Balduini, Erica De CandiaAbstract:The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2 , which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.
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megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome
Platelets, 2015Co-Authors: Luigi Maria Larocca, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Alessandro Pecci, Maria Adele Alberelli, Carlo L. Balduini, Raffaele Landolfi, Nuria Pujolmoix, Marco CattaneoAbstract:The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.
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Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency
Haematologica, 2012Co-Authors: Roberta Bottega, Paula G. Heller, Gian Marco Podda, Nuria Pujol-moix, Ana C. Glembotsky, Alessandro Pecci, Erica De Candia, Patrizia Noris, Daniela De Rocco, Marco CattaneoAbstract:The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.
Jorge Di Paola - One of the best experts on this subject based on the ideXlab platform.
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NBEAL2 mutations and bleeding in patients with gray platelet syndrome.
Platelets, 2018Co-Authors: Fred G Pluthero, Jorge Di Paola, Manuel Carcao, Walter H. A. KahrAbstract:Homozygosity/compound heterozygosity for loss of function mutations in neurobeachin-like 2 (NBEAL2) is causative for Gray platelet syndrome (GPS; MIM #139090), characterized by thrombocytopenia and...
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Spontaneous 8bp Deletion in NBEAL2 Recapitulates the Gray Platelet Syndrome in Mice.
PloS one, 2016Co-Authors: Kärt Tomberg, Rami Khoriaty, Randal J. Westrick, Heather Fairfield, Laura G. Reinholdt, Gary Brodsky, Pavel Davizon-castillo, David Ginsburg, Jorge Di PaolaAbstract:During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the NBEAL2 gene was identified. Though initially considered as a plausible thrombosis modifier, this NBEAL2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the NBEAL2 8 bp deletion (NBEAL2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). NBEAL2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted NBEAL2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.
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De novo 8 bp deletion in the NBEAL2 gene.
2016Co-Authors: Kärt Tomberg, Rami Khoriaty, Randal J. Westrick, Laura G. Reinholdt, Pavel Davizon-castillo, David Ginsburg, Heather E. Fairfield, Gary L. Brodsky, Jorge Di PaolaAbstract:The whole exome sequenced G6-ENU mouse inherited the NBEAL2 deletion from a non-ENU parent 31925 (A). Sanger sequencing validates the heterozygous frameshift mutation in the suppressor pedigree (B). Western blot analysis of washed mouse platelets show a band at the expected size for NBEAL2 (~305kDa) in wildtype mice. This band is missing in NBEAL2tm1Lex/tm1Lex mice as well as mice homozygous for the NBEAL2gps allele (C). Schematic overview of the NBEAL2 gene, the location of the deletion and the expected frameshift (D).
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Emperipolesis of neutrophils in bone marrow and spleen of NBEAL2 deficient mice.
2016Co-Authors: Kärt Tomberg, Rami Khoriaty, Randal J. Westrick, Laura G. Reinholdt, Pavel Davizon-castillo, David Ginsburg, Heather E. Fairfield, Gary L. Brodsky, Jorge Di PaolaAbstract:Increased emperiopolesis of neutrophils (black arrows) in NBEAL2gps/gps mice compared to wildtype was observed in both histologic (A) and cytologic (B) preparations of bone marrow as well as spleen (B and D, respectively).
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expression patterns of NBEAL2 in human tissues and a megakaryocytic cell line
Blood, 2013Co-Authors: Leila Noetzli, Gary Brodsky, Natalie Smith, Jorge Di PaolaAbstract:Abstract Gray platelet syndrome (GPS) is a rare autosomal recessive bleeding disorder characterized by thrombocytopenia, large platelets, and deficient alpha granules in platelets and megakaryocytes. The genetic cause of GPS was recently elucidated by our lab and others, and deleterious mutations were found in the gene NBEAL2 in several affected individuals. NBEAL2 stands for Neurobeachin-like 2 and is a large (30kb, 54 exon) gene on human chromosome 3p21.31. NBEAL2 is a member of a family of proteins that contain a BEACH (Beige and Chediak Higashi) domain which is a highly conserved domain that has been associated with protein functions such as vesicular trafficking, membrane dynamics, and receptor signaling. Very little is known about NBEAL2 other than its involvement with GPS. The relative expression of the NBEAL2 transcript, both in tissues and in subcellular compartments, is not known. It has previously been published that NBEAL2 protein coding isoforms are present in a variety of human tissues, but the abundance of NBEAL2 transcript in each tissue is not known. Furthermore, there is currently no available antibody that recognizes the 302 kDa NBEAL2 protein to validate that mRNA presence translates to protein expression. We have examined the relative transcript abundance of NBEAL2 in a human cDNA library and have validated a novel NBEAL2 antibody in a human megakaryocytic cell line (Dami) and human platelets. We obtained a human mRNA tissue library from Invitrogen which was reverse transcribed to cDNA. Tissues analyzed include bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, bone marrow, peripheral leukocytes, CD33+, and CD36+. To investigate relative transcript abundance, we used a Taqman qPCR probe designed to identify all six protein coding isoforms, as determined by Ensembl ( http://uswest.ensembl.org ). Highest NBEAL2 expression was seen in CD33+ cells, which was 54.3 fold higher than the tissue with lowest expression (skeletal muscle). Relatively high expression was also seen in the peripheral leukocytes, bone marrow, lung, esophagus, and cervix. Alternatively, NBEAL2 expression was low in the brain, despite the homology to the Neurobeachin (NBEA) brain specific protein. While high transcript abundance may infer function, relative protein expression is necessary to validate these findings. Therefore, we designed a novel NBEAL2 antibody against a 14 amino acid peptide (SLEPRRPEEAGAEVC) encoded by exon 1 of NBEAL2 that is 100% conserved between human and mouse. Western blot characterization of this antibody showed the expected approximately 300 kDa band in both soluble and insoluble Dami lysates and human platelet rich plasma lysates. Furthermore, the NBEAL2 antibody was also used for immunofluorescence of Dami cells to determine the approximate subcellular localization of the protein. Preliminary results suggest that NBEAL2 is localized to the cytoplasm, a finding that is consistent with a subcellular localization prediction program (Euk-mPLoc 2.0). In conclusion, we have determined the relative abundance of NBEAL2 transcript in several human tissues, and have begun to characterize a novel antibody against NBEAL2 using the human megakaryocytic Dami cell line and human platelets. Ongoing studies with this novel tool in the NBEAL2 knockout mouse model will likely provide new information about this elusive protein. Disclosures: Di Paola: CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.
Ana C. Glembotsky - One of the best experts on this subject based on the ideXlab platform.
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Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients
Scientific Reports, 2016Co-Authors: Christian A. Di Buduo, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Maria Adele Alberelli, Raffaele Landolfi, Marco Cattaneo, Paola R. Lev, Alessandra Balduini, Erica De CandiaAbstract:The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2 , which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.
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megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome
Platelets, 2015Co-Authors: Luigi Maria Larocca, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Alessandro Pecci, Maria Adele Alberelli, Carlo L. Balduini, Raffaele Landolfi, Nuria Pujolmoix, Marco CattaneoAbstract:The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.
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Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency
Haematologica, 2012Co-Authors: Roberta Bottega, Paula G. Heller, Gian Marco Podda, Nuria Pujol-moix, Ana C. Glembotsky, Alessandro Pecci, Erica De Candia, Patrizia Noris, Daniela De Rocco, Marco CattaneoAbstract:The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.
Gian Marco Podda - One of the best experts on this subject based on the ideXlab platform.
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Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients
Scientific Reports, 2016Co-Authors: Christian A. Di Buduo, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Maria Adele Alberelli, Raffaele Landolfi, Marco Cattaneo, Paola R. Lev, Alessandra Balduini, Erica De CandiaAbstract:The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2 , which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.
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megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome
Platelets, 2015Co-Authors: Luigi Maria Larocca, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Alessandro Pecci, Maria Adele Alberelli, Carlo L. Balduini, Raffaele Landolfi, Nuria Pujolmoix, Marco CattaneoAbstract:The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.
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Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency
Haematologica, 2012Co-Authors: Roberta Bottega, Paula G. Heller, Gian Marco Podda, Nuria Pujol-moix, Ana C. Glembotsky, Alessandro Pecci, Erica De Candia, Patrizia Noris, Daniela De Rocco, Marco CattaneoAbstract:The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.
Paula G. Heller - One of the best experts on this subject based on the ideXlab platform.
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Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients
Scientific Reports, 2016Co-Authors: Christian A. Di Buduo, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Maria Adele Alberelli, Raffaele Landolfi, Marco Cattaneo, Paola R. Lev, Alessandra Balduini, Erica De CandiaAbstract:The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2 , which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.
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megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome
Platelets, 2015Co-Authors: Luigi Maria Larocca, Paula G. Heller, Gian Marco Podda, Ana C. Glembotsky, Alessandro Pecci, Maria Adele Alberelli, Carlo L. Balduini, Raffaele Landolfi, Nuria Pujolmoix, Marco CattaneoAbstract:The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.
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Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency
Haematologica, 2012Co-Authors: Roberta Bottega, Paula G. Heller, Gian Marco Podda, Nuria Pujol-moix, Ana C. Glembotsky, Alessandro Pecci, Erica De Candia, Patrizia Noris, Daniela De Rocco, Marco CattaneoAbstract:The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.
