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.

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

Abstract 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.

scholarly journals Correction of symptoms of platelet storage pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak syndrome

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1196-1201
Author(s):  
EK Novak ◽  
MP McGarry ◽  
RT Swank
Keyword(s):  
Bone Marrow ◽  
Animal Models ◽  
Dense Granule ◽  
Whole Body ◽  
Inherited Disorders ◽  
Humoral Factors ◽  
Storage Pool ◽  
Platelet Storage ◽  
Hermansky Pudlak Syndrome ◽  
Chediak Higashi Syndrome

Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.

Development and Characterization of Anti-Estradiol Polyclonal Antibody

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang
Keyword(s):  
Polyclonal Antibody ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Absorbance ◽  
Standard Curve ◽  
Ic50 Value ◽  
New Zealand White Rabbits

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

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.

Rab38 Expression in Platelet Dense Granule Storage Pool Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2112-2112
Author(s):  
Ivana Ninkovic ◽  
James G. White ◽  
Kenyatta W. Stephens ◽  
Artur Rangel-Fihlo ◽  
Francsisca C. Wu ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Flow Cytometric Analysis ◽  
Dense Granule ◽  
Bleeding Disorder ◽  
Coding Region ◽  
Platelet Dysfunction ◽  
Granule Formation ◽  
Storage Pool ◽  
Storage Pool Disease

Abstract 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.

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 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 Correction of symptoms of platelet storage pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak syndrome

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1196-1201 ◽  
Author(s):  
EK Novak ◽  
MP McGarry ◽  
RT Swank
Keyword(s):  
Bone Marrow ◽  
Animal Models ◽  
Dense Granule ◽  
Whole Body ◽  
Inherited Disorders ◽  
Humoral Factors ◽  
Storage Pool ◽  
Platelet Storage ◽  
Hermansky Pudlak Syndrome ◽  
Chediak Higashi Syndrome

Abstract Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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