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Guy L Reed - One of the best experts on this subject based on the ideXlab platform.
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platelet Dense Granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice
Circulation, 2009Co-Authors: Sarah M King, Rachel A Mcnamee, Aiilyan K Houng, Rakesh P Patel, Michael W Brands, Guy L ReedAbstract:Background— Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet Dense-Granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. Methods and Results— Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3−/−) markedly reduces platelet Dense-Granule secretion. HPS3−/− mice have normal platelet counts, platelet morphology, and α-Granule number, as well as maximal secretion of the α-Granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P<0.05). To examine the role of platelet Dense-Granule secretion in these processes, atherosclerosis-prone mice with combined genetic deficiency of apolipoprotein E and HPS3 (ApoE−/−, HPS3−/−) were compared with congenic, atherosclerosis-prone mice with normal platelet secretion (Apo...
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Platelet Dense-Granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice.
Circulation, 2009Co-Authors: Sarah M King, Rachel A Mcnamee, Aiilyan K Houng, Rakesh P Patel, Michael W Brands, Guy L ReedAbstract:Background— Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet Dense-Granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. Methods and Results— Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3−/−) markedly reduces platelet Dense-Granule secretion. HPS3−/− mice have normal platelet counts, platelet morphology, and α-Granule number, as well as maximal secretion of the α-Granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P
Jean-françois Dubremetz - One of the best experts on this subject based on the ideXlab platform.
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Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon
mBio, 2019Co-Authors: Barbara Fox, Alejandra Falla, Rebekah Guevara, Leah Rommereim, Valéria Bellini, Graciane Petre, Camille Rak, Viviana Cantillana, Jean-françois Dubremetz, Marie-france Cesbron-delauwAbstract:Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized Dense Granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4 mutants was reduced but not abolished. In contrast, the PruΔgra12 mutant was avirulent in mice and PruΔgra12 parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12 and type II PruΔgra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80 and type II PruΔku80 strains, respectively. Despite this resistance, Δgra12 PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated Dense Granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.
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GRA12, a Toxoplasma Dense Granule protein associated with the intravacuolar membranous nanotubular network.
International Journal for Parasitology, 2009Co-Authors: Adeline Michelin, Jean-françois Dubremetz, Corinne Mercier, Amina Bittame, Yann Bordat, Laetitia Travier, Maryse LebrunAbstract:The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named Dense Granules and rhoptries. Here, we describe a new Dense Granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction.
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GRA9, a new Toxoplasma gondii Dense Granule protein associated with the intravacuolar network of tubular membranes.
International journal for parasitology, 2004Co-Authors: Koku D.z. Adjogble, Jean-françois Dubremetz, Marie-france Cesbron-delauw, Corinne Mercier, Christian Hucke, Colin R. Mackenzie, Walter DäubenerAbstract:Important components of the parasitophorous vacuole in which the intracellular protozoan parasite Toxoplasma gondii develops, comprise proteins secreted from apicomplexan specific secretory organelles named the Dense Granules. Here, we confirm by immunofluorescence and by cryo-electron microscopy that the recently isolated B10 protein (318 amino acids, 41kDa) is a new Dense Granule protein that should now be referred to as GRA9. Within the vacuolar compartment, GRA9, like GRA2, GRA4 and GRA6, associates with the network of tubular membranes connected to the parasitophorous vacuole delimiting membrane. Like the other GRA proteins, GRA9 is secreted into the vacuole from the anterior end of the parasite. However, unlike GRA2 or GRA6, GRA9 does not transit by the posterior invaginated pocket of the parasite where the network first assembles. Within the Dense Granules, GRA9 exists in both a soluble and an insoluble state. Like the other GRA proteins, GRA9 is secreted as a soluble form only and like most of the GRA proteins, two forms of GRA9 of the similar molecular weight are detected within the vacuolar space: a soluble form and a membrane associated form. The dual properties of GRA9 are not only ascribed by the presence of amphipathic and hydrophobic alpha-helices but also by the fact that the protein is mainly hydrophilic.
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The expression and distribution of Dense Granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat.
Experimental parasitology, 1999Co-Authors: David J. P. Ferguson, Jean-françois Dubremetz, Marie-france Cesbron-delauw, Keith A. Joiner, L. D. Sibley, S. E. WrightAbstract:The expression and distribution of Dense Granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the Dense Granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large Dense Granules. By immunocytochemistry, Dense Granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which Dense Granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite Dense Granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.
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identification and heterologous expression of a new Dense Granule protein gra7 from toxoplasma gondii
Molecular and Biochemical Parasitology, 1998Co-Authors: D Jacobs, Jean-françois Dubremetz, Anne Loyens, Fons Bosman, Eric SamanAbstract:Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25 857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the Dense Granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new Dense Granule protein GRA7, following the established nomenclature for this protein family.
Marie-france Cesbron-delauw - One of the best experts on this subject based on the ideXlab platform.
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Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon
mBio, 2019Co-Authors: Barbara Fox, Alejandra Falla, Rebekah Guevara, Leah Rommereim, Valéria Bellini, Graciane Petre, Camille Rak, Viviana Cantillana, Jean-françois Dubremetz, Marie-france Cesbron-delauwAbstract:Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized Dense Granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4 mutants was reduced but not abolished. In contrast, the PruΔgra12 mutant was avirulent in mice and PruΔgra12 parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12 and type II PruΔgra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80 and type II PruΔku80 strains, respectively. Despite this resistance, Δgra12 PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated Dense Granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.
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GRA9, a new Toxoplasma gondii Dense Granule protein associated with the intravacuolar network of tubular membranes.
International journal for parasitology, 2004Co-Authors: Koku D.z. Adjogble, Jean-françois Dubremetz, Marie-france Cesbron-delauw, Corinne Mercier, Christian Hucke, Colin R. Mackenzie, Walter DäubenerAbstract:Important components of the parasitophorous vacuole in which the intracellular protozoan parasite Toxoplasma gondii develops, comprise proteins secreted from apicomplexan specific secretory organelles named the Dense Granules. Here, we confirm by immunofluorescence and by cryo-electron microscopy that the recently isolated B10 protein (318 amino acids, 41kDa) is a new Dense Granule protein that should now be referred to as GRA9. Within the vacuolar compartment, GRA9, like GRA2, GRA4 and GRA6, associates with the network of tubular membranes connected to the parasitophorous vacuole delimiting membrane. Like the other GRA proteins, GRA9 is secreted into the vacuole from the anterior end of the parasite. However, unlike GRA2 or GRA6, GRA9 does not transit by the posterior invaginated pocket of the parasite where the network first assembles. Within the Dense Granules, GRA9 exists in both a soluble and an insoluble state. Like the other GRA proteins, GRA9 is secreted as a soluble form only and like most of the GRA proteins, two forms of GRA9 of the similar molecular weight are detected within the vacuolar space: a soluble form and a membrane associated form. The dual properties of GRA9 are not only ascribed by the presence of amphipathic and hydrophobic alpha-helices but also by the fact that the protein is mainly hydrophilic.
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Intracellular trafficking of Dense Granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis.
European journal of cell biology, 1999Co-Authors: Isabelle Coppens, Marie Andries, Jinli L. Liu, Marie-france Cesbron-delauwAbstract:Abstract The Dense Granules of the intracellular protozoan Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole (PV) in which the parasite develops. The biogenesis of Dense Granules as well as the regulatory mechanisms controlling their specific exocytosis are still poorly understood. In this paper, we analyzed the secretory pathway of Dense Granule proteins (GRA proteins) in extracellular T. gondii through the effects of brefeldin A (BFA). Ultrastructural studies of BFA-treated parasites showed disassembly of the Golgi apparatus and accumulation of GRA proteins in a dilated vacuolar system connected to the nuclear envelope. BFA reversibly blocked the intracellular transport of the newly synthesized GRA proteins in a dosedependent manner (blockade of 95 % at 1 μg/ml of BFA). By contrast, discharge of GRA proteins from preformed Dense Granules was unaffected by BFA over a course of 60 min incubation. GRA protein secretion was dependent on incubation temperature as it only occurred above 26°C and it could be stimulated by external factors. This stimulus might be provided by factor(s) present in the serum of the extracellular medium, as incubation of parasites in serum-free medium resulted in a dramatic decrease in protein secretion. Exocytosis can be restored in a dose-dependent fashion by serum addition (maximal stimulatory activity in the 30-200 kDa range) and was optimal at an extracellular pH of 6.5. Altogether, these results demonstrate that GRA proteins are exported through the Golgi apparatus via the classical secretory pathway and can be experimentally discharged from storage Dense Granules as regulated secretory proteins in response to specific stimulation, arguing in favor of a regulated component for Dense Granule exocytosis in T. gondii .
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The expression and distribution of Dense Granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat.
Experimental parasitology, 1999Co-Authors: David J. P. Ferguson, Jean-françois Dubremetz, Marie-france Cesbron-delauw, Keith A. Joiner, L. D. Sibley, S. E. WrightAbstract:The expression and distribution of Dense Granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the Dense Granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large Dense Granules. By immunocytochemistry, Dense Granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which Dense Granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite Dense Granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.
Sarah M King - One of the best experts on this subject based on the ideXlab platform.
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platelet Dense Granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice
Circulation, 2009Co-Authors: Sarah M King, Rachel A Mcnamee, Aiilyan K Houng, Rakesh P Patel, Michael W Brands, Guy L ReedAbstract:Background— Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet Dense-Granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. Methods and Results— Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3−/−) markedly reduces platelet Dense-Granule secretion. HPS3−/− mice have normal platelet counts, platelet morphology, and α-Granule number, as well as maximal secretion of the α-Granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P<0.05). To examine the role of platelet Dense-Granule secretion in these processes, atherosclerosis-prone mice with combined genetic deficiency of apolipoprotein E and HPS3 (ApoE−/−, HPS3−/−) were compared with congenic, atherosclerosis-prone mice with normal platelet secretion (Apo...
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Platelet Dense-Granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice.
Circulation, 2009Co-Authors: Sarah M King, Rachel A Mcnamee, Aiilyan K Houng, Rakesh P Patel, Michael W Brands, Guy L ReedAbstract:Background— Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet Dense-Granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. Methods and Results— Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3−/−) markedly reduces platelet Dense-Granule secretion. HPS3−/− mice have normal platelet counts, platelet morphology, and α-Granule number, as well as maximal secretion of the α-Granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P
James G. White - One of the best experts on this subject based on the ideXlab platform.
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Results of an external proficiency testing exercise on platelet Dense-Granule deficiency testing by whole mount electron microscopy.
American journal of clinical pathology, 2009Co-Authors: Catherine P M Hayward, Karen A Moffat, Elizabeth A Plumhoff, Marnie Timleck, Ernie Spitzer, Sara J. Israels, James G. WhiteAbstract:Performance on specialized diagnostic tests for platelet disorders, including Dense-Granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet Dense-Granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with Dense-Granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and Dense-Granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as Dense Granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.
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The role of Rab38 in platelet Dense Granule defects.
Journal of thrombosis and haemostasis : JTH, 2008Co-Authors: Ivana Ninkovic, James G. White, Artur Rangel-filho, Yvonne H DattaAbstract:Summary. Background: The fawn-hooded hypertensive (FHH) rat has a mutation in the Rab38 gene that is associated with a platelet Dense Granule storage pool disease. Objective: To better characterize the expression and function of Rab38 in FHH rat and human megakaryocytes and platelets. Patients and methods: Rab38 expression in FHH rat and normal tissues was demonstrated by western blotting. Platelet and megakaryocyte morphology and Rab38 expression were examined by transmission electron microscopy and by immunofluorescence confocal microscopy. Platelet surface glycoprotein and P-selectin expression and total serotonin content were assessed by flow cytometry. Results: Rab38 was not expressed in FHH rat tissues, and FHH rat platelets and megakaryocytes lacked Dense Granules. FHH rat platelets had normal expression of surface glycoproteins and of surface P-selectin in response to thrombin. The total serotonin content in FHH rat platelets was similar to that in Brown Norway rat platelets. In a megakaryocyte cell line, Rab38 was expressed in a granular perinuclear and cytoplasmic pattern. There was partial colocalization with serotonin, and minimal colocalization with von Willebrand factor and lysosomal proteins. Conclusions: The lack of Rab38 expression in the FHH rat results in the absence of normal Dense Granules in the megakaryocytes and platelets, which have otherwise normal structure and function. Rab38 may play a role in the development of Dense Granules in the megakaryocytes and platelets.
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rab38 expression in platelet Dense Granule storage pool disease
Blood, 2007Co-Authors: Ivana Ninkovic, James G. White, Kenyatta W Stephens, Artur Rangelfihlo, Yvonne H DattaAbstract:Platelet Dense Granule storage pool disease (SPD) is a bleeding disorder characterized by a lack of normal platelet Dense Granule function, as evidenced by decreased platelet aggregation in response to ADP, epinephrine and collagen. Platelet SPD has been studied most extensively in humans and rodents with Hermansky-Pudlak syndrome (HPS), whose phenotype is a result of defects in Granule trafficking, leading to oculocutanous albinism, lysosomal storage diseases, and platelet dysfunction. We have been characterizing the fawn-hooded hypertensive (FHH) rat, which has been previously shown to have a bleeding disorder consistent with a platelet SPD and some of the features of HPS. While the platelets in the FHH rat have normal alpha Granules and lysosomes, they lack Dense Granules as assessed by transmission electron microscopy. Platelet flow cytometric analysis of GPIb and GPIIb indicated that the FHH platelets have normal surface expression of these adhesion proteins. The FHH rat has a mutation in the Rab38 gene at the ATG start site, which is associated with the bleeding disorder. Rab38 is part of a large family of GTPases, which are involved in Granule formation and secretion. Western blotting of FHH tissues revealed that there is no expression of Rab38 protein. We have used confocal immunomicroscopy to assess Rab38 in platelet formation and function. In normal rat and human platelets, there was punctate expression of Rab38. There was no Rab38 staining detected in FHH platelets. In human megakaryocytic cell lines, Dami and HEL cells, there was punctate staining of Rab38 that was mainly in the periphery of the cells, with a variable amount of perinuclear staining. There was partial colocalization of Rab38 with serotonin and VWF, and with Lamp-3, a marker of lysosomes. The degree of colocalization varied between cells. There was no clear association of Rab38 with actin and tubulin in megakaryocytes. We also examined a cohort of patients with SPD, but not HPS, for mutations in Rab38. The entire coding region and intron-exon boundaries of the Rab38 gene were sequenced in 18 patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Ten of the patients had platelet function defects documented by standard platelet aggregation studies, and eight had no identifiable platelet function defect. No mutations in Rab38 were detected. Whereas numerous known polymorphisms were identified and confirmed, there was no association of any of them with platelet function abnormalities. In conclusion, Rab38 is expressed in platelets and megakaryocytes and may interact with other Granule proteins during megakaryocyte development. Failure to express Rab38 is associated with platelet dysfunction. Further studies are needed to determine its function in megakaryocytes and platelets, and to determine whether defects in Rab38 are a cause of platelet SPD in humans.
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Heterogeneous abnormalities of platelet Dense Granule ultrastructure in 20 patients with congenital storage pool deficiency.
British journal of haematology, 1993Co-Authors: Harvey J. Weiss, Bruce Lages, William J. Vicic, Lancy Y. Tsung, James G. WhiteAbstract:Studies on platelet Dense Granule structure were carried out in 20 patients with various types of congenital storage pool deficiency (SPD), including 15 with specific deficiencies of Dense Granules and Dense Granule substances (delta-SPD), and five with combined deficiencies of Dense and alpha-Granules (alpha delta-SPD). Dense Granules were identified by their high affinity for uranyl ions (uranaffin reaction), by their ability to accumulate the fluorescent dye mepacrine, and by their inherent electron opacity on unfixed, unstained whole mount preparations. By all these methods, Dense Granules were markedly decreased in seven albino patients with the Hermansky-Pudlak syndrome (HPS) variant of delta-SPD. These findings suggest that the basic defect in these patients is a specific abnormality in organelle development which prevents the formation of an intact Granule structure, a quantitative abnormality which may differ from that in animals with related pigment disorders. In contrast, eight non-albino patients with delta-SPD had, on average, only a slightly reduced number of uranaffin-positive and mepacrine-positive Granules, but a shift in uranaffin-Granule distribution towards those lacking a Dense core ('empty Granules'), suggesting a more qualitative type of Dense Granule defect. These results are consistent with previous evidence suggesting a decreased uptake of ATP across the Granule membrane in delta-SPD. In addition, on whole mounts, these patients' platelets contained substantial numbers of electron Dense chains and clusters which contained P and Ca, but with a P/Ca ratio less than that of typical Dense Granules, and which were retained, along with a larger amount of ATP, after thrombin treatment of the platelets. The various findings in these patients raise the possibility that these structures may represent microvesicles, derived from the Golgi apparatus, which provide a transport mechanism for concentrating adenine nucleotides and calcium in Dense Granules and which is impaired in some patients with SPD. Additional defects may account for the more extensive Granule abnormalities observed in alpha delta-SPD.