scholarly journals SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Blood ◽  
2012 ◽  
Vol 120 (2) ◽  
pp. 404-414 ◽  
Author(s):  
Ronghua Meng ◽  
Yuhuan Wang ◽  
Yu Yao ◽  
Zhe Zhang ◽  
Dawn C. Harper ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Subcellular Fractionation ◽  
Dense Granule ◽  
Direct Role ◽  
Excessive Bleeding ◽  
Dense Granules ◽  
Early Endosomes ◽  
Hermansky Pudlak Syndrome ◽  
Endosomal Transport ◽  
Lysosome Related Organelles

Abstract Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

A Functional Basis For The Platelet Storage Pool Deficiency In Chediak-Higashi Cattle

1981 ◽  
Author(s):  
K M Meyers ◽  
M Fukami ◽  
H Holmsen
Keyword(s):  
Freeze Fracture ◽  
Subcellular Fractionation ◽  
Dense Granule ◽  
Bivalent Cation ◽  
Fibrous Matrix ◽  
Dense Granules ◽  
Storage Pool ◽  
Common Basis ◽  
Platelet Storage ◽  
Amine Storage

Platelets from cattle with the morphologic homolog of the Chediak-Higashi (CH) syndrome are essentially devoid of secretable nucleotides and serotonin. There are reduced but still substantial amounts of secretable calcium and magnesium. The storage pool deficiency may be, in part, due to a functional granule defect. Platelets from CH cattle take up serotonin and protect it from degradation for several hours. If these platelets are treated with thrombin, serotonin and bivalent cations are released by mechanisms similar to that of secretion, suggesting a granule location for the released serotonin and cations. This suggestion is verified by subcellular fractionation studies where platelets are first incubated with 14C-serotonin then lysed using a French press. Organelles were then separated on a sucrose gradient by centrifugation. Serotonin in normal bovine platelets is associated with the dense granule or pellet while in CH platelets serotonin is primarily found in a region of the sucrose density zone that in normal platelets contain alpha granules. These findings suggested that some granules in CH platelets are able to acquire the bivalent cation and amine but not the nucleotide component of the bivalent cation-nucleotide-amine storage complex that is thought to occur in normal dense granules.Ultrastructural identification of the serotonin-containing CH granule is not known. There are 2 identifiable granule populations in CH platelets, alpha granules and fibrous matrix granules. Based on serial sectioning freeze fracture and morphometric studies, there are less than 4 of these granules/platelet. Mepacrine studies also demonstrate 2 granule populations. One population has an incidence of 2 per granule and characteristics of normal dense granules. Since the number of fibrous matrix granules and mepacrine granules is similar, a common basis for these granules which has at least some dense granule characteristics, i.e., mepacrine storage, is suggested.

scholarly journals Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JM Gerrard ◽  
D Lint ◽  
PJ Sims ◽  
T Wiedmer ◽  
RD Fugate ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein S ◽  
Dense Granule ◽  
Western Blots ◽  
Dense Granules ◽  
Granule Membrane ◽  
Thrombin Activation ◽  
Hermansky Pudlak Syndrome ◽  
Slot Blot Analysis

Abstract Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

The pink gene encodes the Drosophila orthologue of the human Hermansky–Pudlak syndrome 5 (HPS5) gene

Genome ◽  
2007 ◽  
Vol 50 (6) ◽  
pp. 548-556 ◽  
Author(s):  
Monika Syrzycka ◽  
Lori A. McEachern ◽  
Jennifer Kinneard ◽  
Kristel Prabhu ◽  
Kathleen Fitzpatrick ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Protein Complexes ◽  
Model Organism ◽  
Autosomal Recessive Disorders ◽  
Dense Granules ◽  
Eye Colour ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
Key Participants

Hermansky–Pudlak syndrome (HPS) consists of a set of human autosomal recessive disorders, with symptoms resulting from defects in genes required for protein trafficking in lysosome-related organelles such as melanosomes and platelet dense granules. A number of human HPS genes and rodent orthologues have been identified whose protein products are key components of 1 of 4 different protein complexes (AP-3 or BLOC-1, -2, and -3) that are key participants in the process. Drosophila melanogaster has been a key model organism in demonstrating the in vivo significance of many genes involved in protein trafficking pathways; for example, mutations in the “granule group” genes lead to changes in eye colour arising from improper protein trafficking to pigment granules in the developing eye. An examination of the chromosomal positioning of Drosophila HPS gene orthologues suggested that CG9770, the Drosophila HPS5 orthologue, might correspond to the pink locus. Here we confirm this gene assignment, making pink the first eye colour gene in flies to be identified as a BLOC complex gene.

scholarly journals Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JM Gerrard ◽  
D Lint ◽  
PJ Sims ◽  
T Wiedmer ◽  
RD Fugate ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein S ◽  
Dense Granule ◽  
Western Blots ◽  
Dense Granules ◽  
Granule Membrane ◽  
Thrombin Activation ◽  
Hermansky Pudlak Syndrome ◽  
Slot Blot Analysis

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

Incomplete Platelet Dense Granule Formation in Normal Neonates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3210-3210
Author(s):  
Walter H. Kahr ◽  
Shoma Baidya ◽  
Animitra Das ◽  
Ayca Toprak ◽  
Hilary Christensen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Thin Section ◽  
Platelet Activation ◽  
Platelet Function ◽  
Blood Platelets ◽  
Coagulation Factor ◽  
Dense Granule ◽  
Older Children ◽  
Granule Formation ◽  
Dense Granules

Abstract Platelets are important in maintaining hemostasis in newborns, where bleeding can arise from abnormal platelet function and/or thrombocytopenia. It is well established that plasma coagulation factor concentrations are lower in neonates compared to children and adults, but less is known about the development and function of neonatal platelets. It has been postulated that platelets from neonates, and to a greater extend from premature neonates, are dysfunctional due to low dense granule counts (Blood2006;108:331a), however, other studies have shown normal neonate platelet function. Our previous studies indicated a slightly decreased number of dense granules per platelet in neonates (Blood2005;106:4159–4156). We have now extended these studies to a larger cohort of 19 normal neonatal cord blood samples (gestational age 37.5–40 weeks) from planned Caesarean sections, which were analyzed under optimal sample handling conditions and compared to platelets from 10 children (age 8–10 years). 50 platelets from each subject were evaluated for dense granule content utilizing whole mount and thin section electron microscopy (EM) for the quantification of dense granules (detected via their electron-dense calcium content) and ultrastructural assessment. A subset of samples was tested via flow cytometry for P-selectin expression as a measure of platelet activation, and platelet structural integrity was also assessed using thin section EM. Our data revealed that platelets in neonatal cord blood had a mean dense granule count of 2.3 (SD=2.2) per platelet, compared to 4.4 dense granules per platelet (SD=2.7) in blood from older children; t-test comparisons showed the difference between these groups to be highly significant (P<0.001). Interestingly, 22% of cord blood platelets contained no measurable dense granules, whereas only 3% of platelets from older children where devoid of dense granules. We suspected that the mean dense granule counts of <1 per platelet in neonatal cord blood reported by others may have arisen due to high levels of platelet activation during sample acquisition or handling. In our samples platelet activation as measured by P-selectin expression was similar in both populations and did not exceed 7.5%, and platelet morphology as assessed by thin section EM was also comparable. Our studies confirm that neonatal cord blood platelets contain fewer recognizable dense granules than those found in older children. Two possible explanations for this observation are: normal numbers of dense granules are present in neonatal platelets, but a subset cannot be detected via EM owing to insufficient calcium uptake; there are fewer dense granules in neonatal platelets owing to peculiarities in the development of megakaryocytes, where recent studies have suggested that dense granules originate by an active transport mechanism and move into proplatelets. These possibilities point to the usefulness of studying fetal and neonatal megakaryopoiesis.

scholarly journals Sandy: a new mouse model for platelet storage pool deficiency

1991 ◽  
Vol 58 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Richard T. Swank ◽  
Hope O. Sweet ◽  
Muriel T. Davisson ◽  
Madonna Reddington ◽  
Edward K. Novak
Keyword(s):  
Dense Granule ◽  
Recessive Mutation ◽  
Mouse Mutant ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage ◽  
Autosomal Recessive Mutation ◽  
Acidification Activity ◽  
Hermansky Pudlak Syndrome ◽  
Reduced Rate

SummarySandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near thecrandXtgenetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.

scholarly journals The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1533-1540 ◽  
Author(s):  
Sreenivasulu Chintala ◽  
Jian Tan ◽  
Rashi Gautam ◽  
Michael E. Rusiniak ◽  
Xiaoli Guo ◽  
...  
Keyword(s):  
Positional Cloning ◽  
Adenine Nucleotides ◽  
Dense Granule ◽  
Sugar Transporter ◽  
Nucleotide Sugar ◽  
Dense Granules ◽  
Transporter Family ◽  
Chediak Higashi Syndrome ◽  
Lysosome Related Organelles ◽  
Nucleotide Sugar Transporter

Abstract Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency.

A New Gene Which Regulates Platelet Dense Granules.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2177-2177
Author(s):  
Rashi Gautam ◽  
Michael E. Rusiniak ◽  
Sreenivasulu Chintala ◽  
Jian Tan ◽  
Wei Li ◽  
...  
Keyword(s):  
Coat Color ◽  
Single Gene ◽  
Backcross Progeny ◽  
Genomic Region ◽  
Dense Granule ◽  
Dense Granules ◽  
Prolonged Bleeding ◽  
Platelet Storage ◽  
New Gene ◽  
Lysosome Related Organelles

Abstract Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease characterized by defects in trafficking of intracellular vesicles to lysosome-related organelles such as melanosomes, lung lamellar bodies and platelet dense granules. The ashen (rab27a) mouse coat color mutant maintained at Roswell Park Cancer Institute (ash-Roswell) has been proposed as an animal model of HPS based upon prolonged bleeding times, decreased platelet aggregation and greatly decreased platelet dense granule components, including serotonin and ADP. However, human patients with Rab27a deficiency (Griscelli Syndrome) and other isolates of mouse ashen mutants do not exhibit prolonged bleeding suggesting an additional mutation in a gene affecting platelets but not melanocytes in the ash-Roswell mouse. In order to identify this mutation, a backcross between ash-Roswell and the PWK control strain was analyzed. In 390 backcross progeny, deficient platelet serotonin and diluted coat color (Rab27a) phenotypes segregated independently and in the proportions expected for single gene traits. The gene for serotonin deficiency was narrowed to a 2.1Mb region of mouse chromosome 10 containing 15 genes. None of these genes have been implicated previously in platelet storage pool deficiency. These studies indicate this genomic region contains a gene which uniquely controls platelet dense granules and is a candidate gene for a new form of human HPS.

scholarly journals BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

2007 ◽  
Vol 18 (3) ◽  
pp. 768-780 ◽  
Author(s):  
Subba Rao Gangi Setty ◽  
Danièle Tenza ◽  
Steven T. Truschel ◽  
Evelyn Chou ◽  
Elena V. Sviderskaya ◽  
...  
Keyword(s):  
Protein Complex ◽  
Genetic Disorder ◽  
Adaptor Protein ◽  
Transport Pathways ◽  
Cargo Transport ◽  
Early Endosomes ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
And Function ◽  
Tyrosinase Related Protein

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1–deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.

scholarly journals BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

2016 ◽  
Vol 214 (3) ◽  
pp. 293-308 ◽  
Author(s):  
Megan K. Dennis ◽  
Cédric Delevoye ◽  
Amanda Acosta-Ruiz ◽  
Ilse Hurbain ◽  
Maryse Romao ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Rab Gtpase ◽  
Cargo Delivery ◽  
Cargo Protein ◽  
Early Endosomes ◽  
Tubular Transport ◽  
Hermansky Pudlak Syndrome ◽  
Recycling Pathway ◽  
Lysosome Related Organelles ◽  
Dependent Transport

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants.

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