A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents

Summary. Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and α-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 μl of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 μl of this cell suspension were incubated with mepacrinc alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 μl of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.     

Giant platelet granules in a child with the Chediak-Higashi syndrome.

Previous ultrastructural investigation have not identified abnormal lysosomes in platelets obtained from humans or animals with the Chediak-Higashi Syndrome. We report here a patient whose megakaryocytes and platelets were found to contain giant granules when viewed by light and electron microscopy. The granules measured up to 1.5 micrometer in diameter, contained either homogeneous or heterogeneous material, were acid phosphatase positive, and were present in approximately 30% of bone marrow megakaryocytes and 5% of circulating platelets. A decrease was observed in serotonin containing dense granules, serotonin uptake and serotonin release as reported previously. Microtubules in platelets and megakaryocytes were intact and no other morphologic abnormalities were identified. No clinical evidence of bleeding was observed in this patient and platelet counts have been normal. The lack of giant platelet lysosomes in other reported cases of Chediak-Higashi Syndrome attests to significant heterogeneity in this disease with a spectrum of clinical and laboratory findings.     

A flow cytometric assay for the study of dense granule storage and release in human platelets

The clinical manifestations of platelet dense (δ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83±6 (mean ± 1 SD, range 69–91). The difference in MFI between resting and stimulated platelets was 28±7 (range 17–40). Six members of a family, of whom one had a known δ-storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.     

A flow cytometric assay for the study of dense granule storage and release in human platelets

The clinical manifestations of platelet dense ( delta ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83+/-6 (mean +/- 1 SD, range 69-91). The difference in MFI between resting and stimulated platelets was 28+/-7 (range 17-40). Six members of a family, of whom one had a known delta -storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.     

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

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.     

Platelet storage pool deficiency in mouse pigment mutations associated with seven distinct genetic loci

Seven mouse pigment mutants, which have alterations at distinct genes, are known to have a defect in kidney lysosomal enzyme secretion. Two of these, beige and pale ear, have a bleeding abnormality associated with a deficiency in the number of platelet dense granules. In the present study, five other mutants with defective lysosomal enzyme secretion-- pearl, pallid, light ear, maroon, and ruby-eye--were likewise found to have abnormally prolonged bleeding times after experimental injury. Platelet counts were similar to those of normal mice, but the platelet dense granule components serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) and morphologically identifiable dense granules were markedly reduced in these mutants. The capacity to accumulate exogenous 3H-serotonin in platelets was reduced 2-3-fold. Thrombin-stimulated secretion of 3H-serotonin was slightly decreased in all mutants. However, the seven mutants could be subdivided into three groups based on the degree of secretion of lysosomal enzymes after thrombin stimulation. Thus, all seven mouse pigment mutants have symptoms consistent with platelet storage pool deficiency and may serve as useful animal models for specific types of human platelet storage pool disease. Also, the results emphasize the genetic, morphological, and functional interrelatedness of three organelles: melanosomes, lysosomes, and platelet dense granules.     

Cocoa: a new mouse model for platelet storage pool deficiency

We describe genetic, haematological and biochemical properties of a new mouse pigment mutant, cocoa (coa). Cocoa is a recessive mutation located on the centromeric end of chromosome 3 near the Car-2 locus. The mutation causes increased bleeding time accompanied by symptoms of platelet storage pool deficiency (SPD), including decreased platelet serotonin and decreased visibility of dense granules as analysed by electron microscopy of unfixed platelets. Dense granules were visible in normal numbers when platelets were incubated with the fluorescent dye, mepacrine. The intragranular environment, however, was abnormal as indicated by decreased flashing of mepacrine-loaded dense granules after exposure to ultraviolet light. Unlike the previously described seven mouse pigment mutations with SPD in which pigment granules, platelet dense granules and lysosomes are affected, the cocoa mutant had normal secretion of lysosomal enzymes from kidney proximal tubule cells and platelets. The cocoa mutation thus represents an example of a single gene which simultaneously affects melanosomes and platelet dense granules but probably does not affect lysosomes. The results indicate that melanosomes and platelet dense granules share steps in synthesis and/or processing. Cocoa may be a model for cases of human Hermansky-Pudlak syndrome in which functions of melanosomes and platelet dense granules, but not lysosomes, are involved.     

The mouse pale ear pigment mutant as a possible animal model for human platelet storage pool deficiency

The mouse pigment mutant pale ear, ep/ep, which has a defect in kidney lysosomal enzyme secretion, had prolonged bleeding on experimental injury. Platelet counts and platelet protein did not differ from normal. There was, however, a deficiency in the platelet dense granule contents, serotonin, ATP, and ADP. Furthermore, a marked reduction of platelet dense granules was observed by electron microscopy. The results suggest that pale ear is a useful animal model in the study of platelet storage pool disease. Studies on this mutant and other pigment mutants have established that one gene can regulate at least three subcellular organelles, including the melanosome, the lysosome, and the platelet dense granule.     

Gray platelet syndrome: immunoelectron microscopic localization of fibrinogen and von Willebrand factor in platelets and megakaryocytes

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha- granule content.     

The regulation of platelet-dense granules by Rab27a in the ashen mouse,a model of Hermansky-Pudlak and Griscelli syndromes,is granule-specific and dependent on genetic background

The ashen (ash) mouse, a model for Hermansky-Pudlak syndrome (HPS) and for a subset of patients with Griscelli syndrome, presents with hypopigmentation, prolonged bleeding times, and platelet storage pool deficiency due to a mutation which abrogates expression of the Rab27a protein. Platelets of mice with the ashen mutation on the C3H/HeSnJ inbred strain background have greatly reduced amounts of dense granule components such as serotonin and adenine nucleotides though near-normal numbers of dense granules as enumerated by the dense granule-specific fluorescent dye mepacrine. Thus, essentially normal numbers of platelet dense granules are produced but the granule interiors are abnormal. Collagen-mediated aggregation of mutant platelets is significantly depressed. No abnormalities in the concentrations or secretory rates of 2 other major platelet granules, lysosomes and alpha granules, were apparent. Similarly, no platelet ultrastructural alterations other than those involving dense granules were detected. Therefore, Rab27a regulates the synthesis and secretion of only one major platelet organelle, the dense granule. There were likewise no mutant effects on levels or secretion of lysosomal enzymes of several other tissues. Together with other recent analyses of the ashen mouse, these results suggest a close relationship between platelet dense granules, melanosomes of melanocytes and secretory lysosomes of cytotoxic T lymphocytes, all mediated by Rab27a. Surprisingly, the effects of the ashen mutation on platelet-dense granule components, platelet aggregation, and bleeding times were highly dependent on genetic background. This suggests that bleeding tendencies may likewise vary among patients with Griscelli syndrome and HPS with Rab27a mutations.     

Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha

Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.     

Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency

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 (δ-SPD), and five with combined deficiencies of dense and α-granules (αδ-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 δ-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 δ-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 δ-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 αδ-SPD.     

Incorporation of a circulating protein into megakaryocyte and platelet granules.

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.     

The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole-mount and quinacrine-fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky-Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.     

Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice

Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG-/- mice. Wild-type (WT) but not SG-/- platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal alpha-granule structure in SG-/- platelets. However, SG-/- platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG-/- megakaryocytes showed extensive emperipolesis of neutrophils. SG-/- platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG-/- platelets. The alpha-granule proteins platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG-/- platelets. Exposure of P-selectin and alphaIIb after thrombin treatment was similar in WT and SG-/- platelets. SG-/- mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG-/- platelet function deficiencies are related to inadequate packaging and secretion of selected alpha-granule proteins and reduced secretion of dense granule contents critical for platelet activation.     

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

Platelet proteome and function in X−linked thrombocytopenia with thalassemia and in silico comparisons with gray platelet syndrome

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a b-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet a- and dense granules. The proteomes of isolated blood platelets from five male XLTT patients, compared to five sex- and agematched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change ≥±1.2, q<0.05). Of 46 downregulated proteins, 39 were previously reported to be associated with platelet granules. Reduced protein levels of PTGS1 and SLC35D3 were validated in megakaryocytes of XLTT bone marrow biopsies by immunohistochemistry. Platelet function testing by flow cytometry revealed low dense- and a-granule release and fibrinogen binding in response to ligation of receptors for ADP, the thrombin receptor PAR4 and the collagen receptor GPVI. Significant reductions of a number of agranule proteins overlapped with a previous platelet proteomics investigation in the inherited macrothrombocytopenia gray platelet syndrome. In contrast, Ca²⁺ transporter proteins that facilitate dense granule release were downregulated in XLTT but upregulated in gray platelet syndrome. Ingenuity pathway analysis showed altered coagulation system and protein ubiquitination pathways in the XLTT platelets. Collectively, the results revealed protein and functional alterations affecting platelet a- and dense granules in XLTT, probably contributing to bleeding.     

In vivo biotinylation studies: specificity of labelling of reticulated platelets by thiazole orange and mepacrine

Animal in vivo biotinylation studies have demonstrated that thiazole orange (TO) labels the youngest cells in the circulation. TO has since been widely used for the measurement of reticulated platelets. As recent findings suggest that at high concentrations TO also labels platelet dense granules non-specifically, the value of previous work is unclear. Mepacrine also labels platelet dense granules and can detect storage pool defects. In this study, a mouse in vivo biotinylation model was used to determine the specificity of TO and mepacrine staining on platelets recently released into the circulation. The mean life span of biotin/TO (low), biotin/TO (high) and mepacrine/TO dual-positive platelets was 1.4 d (SD 0.5), 2.2 d (SD 0.2) and 2.3 d (SD 0.3) respectively (n = 6) compared with a life span for biotin-positive platelets of 4.9 d (SD 1.6). TO (low), TO (high) and mepacrine labelled 8.0% (SD 3.1), 43.9% (SD 8.3) and 40.0% (SD 9.9) of the total platelet population respectively (results of 30 samples from six mice), which decreased to 6.8% (SD 3. 9), 26.6% (SD 6.9) and 25.7% (SD 10.6) after thrombin degranulation. The shorter life span and lack of thrombin sensitivity of TO (low)-positive platelets, suggests that TO (low) measures reticulated platelets specifically. The comparative life spans and thrombin sensitivity of TO (high) and mepacrine-positive platelets suggest that TO (high) labels platelet dense granules. These data also suggest that dense granules are lost during platelet ageing.     

Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal

Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP- binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP- binding proteins may affect one or both processes.