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.

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.

scholarly journals Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 143-152 ◽  
Author(s):  
SJ Israels ◽  
JM Gerrard ◽  
YV Jacques ◽  
A McNicol ◽  
B Cham ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Serotonin Release ◽  
Dense Granule ◽  
Double Labeling ◽  
Platelet Surface ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Dense Granule Secretion

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

The Lysosomal Granule Membrane Protein, Lamp-2, Is also Present in Platelet Dense Granule Membranes

1996 ◽  
Vol 75 (04) ◽  
pp. 623-629 ◽  
Author(s):  
S J Israels ◽  
E M McMillan ◽  
C Robertson ◽  
S Singhroy ◽  
A McNicol
Keyword(s):  
Membrane Protein ◽  
Cell Types ◽  
Surface Expression ◽  
Dense Granule ◽  
Similar Distribution ◽  
Dense Granules ◽  
Granule Membrane ◽  
Early Expression ◽  
Late Expression ◽  
Granule Release

SummaryLysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.

scholarly journals Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 143-152 ◽  
Author(s):  
SJ Israels ◽  
JM Gerrard ◽  
YV Jacques ◽  
A McNicol ◽  
B Cham ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Serotonin Release ◽  
Dense Granule ◽  
Double Labeling ◽  
Platelet Surface ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Dense Granule Secretion

Abstract We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

scholarly journals Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1231-1237 ◽  
Author(s):  
A Shalev ◽  
G Michaud ◽  
SJ Israels ◽  
A McNicol ◽  
S Singhroy ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Dense Granule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Granule Formation ◽  
Standard Curve ◽  
Storage Pool ◽  
Granule Membrane ◽  
Hermansky Pudlak Syndrome ◽  
Novel Protein

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

scholarly journals Platelet and Megakaryocyte Dense Granules Contain Glycoproteins Ib and IIb-IIIa

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 4047-4057 ◽  
Author(s):  
Tayebeh Youssefian ◽  
Jean-Marc Massé ◽  
Francine Rendu ◽  
Josette Guichard ◽  
Elisabeth M. Cramer
Keyword(s):  
Plasma Membrane ◽  
Dense Body ◽  
Secretory Granules ◽  
Dense Granule ◽  
Immunogold Labeling ◽  
Membrane Receptors ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Plasma Membrane Receptors

Abstract Platelets contain two main types of secretory organelles, the dense granules and the α-granules. P-selectin, a specific receptor for leukocytes that is present in the α-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the α-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti–Gp IIb-IIIa or anti–Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti–Gp IIb-IIIa or anti–Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additionnal evidence of similarities between the α-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of α-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.

scholarly journals TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+release

2015 ◽  
Vol 26 (18) ◽  
pp. 3263-3274 ◽  
Author(s):  
Andrea L. Ambrosio ◽  
Judith A. Boyle ◽  
Santiago M. Di Pietro
Keyword(s):  
Dense Granule ◽  
Membrane Dynamics ◽  
Vesicular Trafficking ◽  
Pore Channel ◽  
Calcium Stores ◽  
Dense Granules ◽  
Granule Membrane ◽  
Specific Proteins ◽  
Normal Hemostasis ◽  
First Time

Platelet dense granules (PDGs) are acidic calcium stores essential for normal hemostasis. They develop from late endosomal compartments upon receiving PDG-specific proteins through vesicular trafficking, but their maturation process is not well understood. Here we show that two-pore channel 2 (TPC2) is a component of the PDG membrane that regulates PDG luminal pH and the pool of releasable Ca2+. Using a genetically encoded Ca2+biosensor and a pore mutant TPC2, we establish the function of TPC2 in Ca2+release from PDGs and the formation of perigranular Ca2+nanodomains. For the first time, Ca2+spikes around PDGs—or any organelle of the endolysosome family—are visualized in real time and revealed to precisely mark organelle “kiss-and-run” events. Further, the presence of membranous tubules transiently connecting PDGs is revealed and shown to be dramatically enhanced by TPC2 in a mechanism that requires ion flux through TPC2. “Kiss-and-run” events and tubule connections mediate transfer of membrane proteins and luminal content between PDGs. The results show that PDGs use previously unknown mechanisms of membrane dynamics and content exchange that are regulated by TPC2.

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.

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.

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