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 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.

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.

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.

Identification of Dense Granule Specific Membrane Proteins in Bovine Platelets that Are Absent in the Chediak-Higashi Syndrome

1990 ◽  
Vol 64 (02) ◽  
pp. 319-325 ◽  
Author(s):  
Kenneth Meyers ◽  
Carrie Seachord
Keyword(s):  
Membrane Proteins ◽  
Dense Granule ◽  
Dense Granules ◽  
Granule Fraction ◽  
Granule Membrane ◽  
Alternative Hypotheses ◽  
Hypotonic Lysis ◽  
Chediak Higashi Syndrome ◽  
Granule Membrane Proteins ◽  
Specific Membrane

SummaryPlatelets from cattle with the Chediak-Higashi syndrome (CHS) have a platelet dense granule deficiency. One hypothesis for the platelet dense granule deficiency is that the granule is simply not formed in CHS megakaryocytes (MK). Alternative hypotheses include that the granule is assembled in CHS MK but a functional amino-nucleotide-cation storage complex cannot be formed or that the dense granule or its precursor fuses with other granules. This study was undertaken to determine if membrane proteins specific for platelet dense granules can be identified in membranes of other granules in CHS platelets. Platelets were disrupted; a mixed-granule fraction and alpha-granule enriched, mitochondrial-enriched, and dense granule-enriched subfractions were obtained. Membrane proteins in these intact granules were radiolabeled and the granule underwent hypotonic lysis. Membrane proteins were extracted from granule “ghosts”, separated, and then visualized by autoradiography. Three major proteins were identified in platelet dense granule membrane subfractions. Two of these proteins could be identified in membrane extracts from the mixed-granule fraction from normal platelets. They could neither be identified in extracts from the mixed granule fraction of CHS platelets nor in membranes from alpha granule-enriched and mitochondrial-enriched subfractions. The absence of dense granule membrane proteins in membranes of other organelles within CHS platelets suggests that fusion of dense granules or its precursor with other granules cannot account for the platelet dense granule deficiency in CHS platelets.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

The Appearance Of PF1 And PF3 In Activated Human Platelets

1981 ◽  
Author(s):  
H Sandberg ◽  
A P Bodet ◽  
F A Dombrosei ◽  
L O Andersson ◽  
B R Lentz
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Void Volume ◽  
Factor V ◽  
Dense Granules ◽  
Protein Particles ◽  
Hydrolysis Of ◽  
Platelet Pellet ◽  
Stimulation Of

Collagen and thrombin induced platelet activation were examined, in vitro, with regard to the appearance of surface-associated Factor V-like activity (PF1) and catalytic phospholipid-like surface activity (PF3). Two test systems were used: a clotting assay (a modified KAPTT) and a chromogenic substrate assay (maximum hydrolysis of S-2238). Following stimulation of normal platelets, both PF1 and PF3 appeared simultaneously in the supernatant and platelet pellet. When normal platelets were collected and carefully washed in a buffer containing adenosine, PGE1, and theophylline, the appearance of both PF1 and PF3 was blocked, as was the release of ATP from dense granules, the release of β-TG and PF4 from α-granules, and the occurrence of aggregation. When platelets were collected in this same inhibitor-containing buffer, and then gel filtered/centrifuge-washed in an inhibitor-free buffer, the appearance of PF1 and PF3 was still blocked. This occurred even though release of ATP, β-TG and PF4 as well as aggregation followed a pattern equivalent to platelets never exposed to these inhibitors. When the release supernatant from normal platelets isolated in the absence of inhibitors was gel filtered on Sepharose CL-4B in the presence of EDTA, the carbohydrate-free, lipid- protein particles (70-170nm diam.) that provide PF3 appeared in the void volume. When the release supernatant from normal platelets was gel filtered in the presence of Ca2+, both, PF1 and PF3 eluted in the void volume. With platelets isolated from severe F.V-deficient donors, only PF3 was found in the void volume, in the presence or absence of Ca2+. It seems that the appearance of PF1 and PF3 as coagulant activities is completely separate from both the release of dense granule and α-granule contents as well as platelet aggregation and that the appearance of PF1 requires the presence of Ca2+.

scholarly journals Immature dense granules in platelets from mice with platelet storage pool disease

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1300-1306 ◽  
Author(s):  
M Reddington ◽  
EK Novak ◽  
E Hurley ◽  
C Medda ◽  
MP McGarry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dense Granule ◽  
Mutant Mice ◽  
Normal Numbers ◽  
Group Of Seven ◽  
Dense Granules ◽  
Storage Pool ◽  
Platelet Storage ◽  
Transmission Electron ◽  
Storage Pool Disease

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.

Sign in / Sign up

Export Citation Format

Share Document