The storage pool deficiency in platelets from humans with the Chédiak-Higashi syndrome: study of six patients

Functional and biochemical studies of platelets from human Chédiak-Higashi syndrome (CHS) are scarce and/or incomplete. In the present report, the aggregation response to a variety of inducers of platelet aggregation, the content of the dense granule constituents ATP, ADP, serotonin and calcium, the secretion of ATP, ADP, and calcium induced by thrombin, the total content of magnesium, the incorporation of 14C-adenine in the cytoplasmic pool of adenine nucleotides, as well as the content of intracellular cyclic-AMP, have been quantitated in six patients with CHS. Furthermore, data is presented on the kinetics of uptake of radiolabelled serotonin and its storage in human CHS platelets. An abnormal aggregation behaviour was found in all patients. However, the response of CHS platelets to the different inducers studied did not show a uniform pattern. The total content and the maximal amounts of the dense granule constituents secretable by thrombin were greatly decreased in all six patients. Total magnesium content was similar to that of normal platelets. The ATP/ADP ratio was higher than in controls. Uptake of radiolabelled serotonin by CHS platelets closely followed the uptake by normal platelets; during the first 2-3 min, however, incorporation of the amine by CHS platelets came rapidly to a plateau which contrasts with the steady, linear increase in uptake found in controls. CHS platelets loaded with radiolabelled serotonin and gel-filtered, showed a spontaneous release of radioactivity not observed in normal platelets under the same conditions. The cyclic-AMP content of CHS platelets was similar to that of normals. In contrast to platelets from patients with storage pool disease, the secretable calcium from CHS platelets represents a 67% of total platelet calcium (61% in normals), suggesting that the absolute values for the non-secretable portion in CHS platelets must be very low. The results reported confirm the existence of a true storage pool deficiency of the dense granule constituents as a common defect in CHS platelets. The variety of responses among patients, to the different aggregatory stimuli studied, can not be solely ascribed to the storage pool deficiency described.     

Decreased nucleotide and serotonin storage associated with defective function in Chediak-Higashi syndrome cattle and human platelets

Prolonged mean template bleeding time of 14 min observed in seven cattle with the Chediak-Higashi syndrome (CHS) prompted the examination of platelet function in these animals. There was no distinguishable difference in concentration of circulating platelets between CHS and control cattle. CHS bovine platelets failed to aggregate in vitro in response to concentrations of acid-soluble collagen which induced aggregation in all normal samples. The primary platelet response to 5 muM ADP was normal in CHS cattle. A markedly decreased amount of serotonin (1.2% of normal) was detected in CHS bovine platelets. Bovine CHS platelet ATP and ADP contents were significantly less than normal, and the ATP/ADP ratios were 5.04 in normal and 29.38 in CHS platelets. Results of these animal investigations prompted a similar study of two patients with CHS. In humans, an increased bleeding time greater than 15 min and an in vitro impaired aggregation response to acid-soluble collagen and 5 muM adrenaline were discovered. Both ATP and ADP were reduced in CHS human platelets, and the ATP/ADP ratio was 3.96, compared to a ratio of 1.52 for platelets of two normal subjects. These findings suggested the presence of a "storage pool disease" of CHS platelets.     

Storage pool deficiency in cattle with the Chediak-Higashi syndrome results from an absence of dense granule precursors in their megakaryocytes

Platelets from cattle with the Chediak-Higashi syndrome (CHS) have a storage pool deficiency and virtual absence of platelet dense granules. Megakaryocytes (MKs) from five control (n = 135) and five CHS (n = 133) cattle were evaluated using standard transmission electron microscopy. Osmiophilic dense granules were not observed in control or CHS MKs. In MKs from normal cattle, clear vesicles of 200- to 650-nm diameter bounded by a sharp membrane were observed. They were easily differentiated from the demarcation membrane system, endoplasmic reticulum, and alpha granules. The clear vesicles were virtually absent in MKs from CHS cattle at all stages of maturation. MKs in bone marrow samples from two control (n = 91) and two CHS (n = 61) cattle that had been processed for the uranaffin reaction were also evaluated. The clear vesicles were replaced by uranaffin-positive granules in MKs from control cattle, but positive uranaffin granules were not observed in CHS MKs. These findings indicate that the platelet dense granule storage pool deficiency in CHS cattle results from an anatomic absence of dense granule precursors in maturing and mature CHS MKs.     

Selective decrease in platelet dense granule adenine nucleotides during recovery from acute experimental thrombocytopenia and ensuing thrombocytosis in baboons

Serial measurements of platelet volume, platelet content of adenine nucleotides, beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and ex vivo platelet aggregation were made in baboons under basal, steady-state conditions of normal platelet production, and during recovery from acute thrombocytopenia induced by the exposure of flowing blood to spherical glass microbeads. The mean basal platelet count of 509 +/- 107 X 10(9)/l (+/- 1 SD; n = 4) fell acutely to 36.8 +/- 12.6 X 10(9)/l after the insertion of glass bead columns and blood filters, placed distally, for 60 min into surgically implanted arteriovenous-shunts in heparinized baboons. After the irreversible removal of up to 90% of the baseline circulating platelet population, recovery from thrombocytopenia was characterized by a constant rate of increase in circulating platelet counts (115 +/- 11 X 10(9)/l/d) and a rebound thrombocytosis to 1.5 times the basal platelet count after 7 d. Steadystate thrombocytopoiesis was achieved by 3-4 weeks after the onset of thrombocytopenia. Platelet dense granule ADP and ATP decreased significantly from 3.89 +/- 0.20 and 2.33 +/- 0.25 mumol/10(11) platelets respectively at baseline to 2.17 +/- 0.37 and 1.68 +/- 0.37 mumol/10(11) platelets respectively after 7 d (P less than 0.001 in both cases) and normalization was achieved only after 4 weeks. By contrast, the mean platelet volume and platelet content of beta-TG and PF4 did not change significantly throughout the course of study (P greater than 0.1 in both cases). Platelet function, assessed by platelet aggregation ex vivo, demonstrated that platelet function was not impaired despite the significant decrease in dense granule ADP. We conclude that a selective, temporal reduction in platelet dense granule adenine nucleotides reflects changes in the thrombocytopoietic control mechanism secondary to induction of acute thrombocytopenia.     

Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD 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.     

Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules

Patients whose platelets are deficient in glycoprotein (GP) Ib, IIb- IIIa (thrombasthenia), or granule substances (storage pool deficiency, SPD) were studied to define further the properties of platelets that mediate platelet adhesion and thrombus formation on subendothelium. Both nonanticoagulated and citrated blood were exposed to everted, de- endothelialized rabbit vessel segments under controlled flow conditions and shear rates varying from 650 to 3,300 sec-1. Morphometry was used to measure platelet thrombus dimensions and the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets. Adhesion was defined as C + S. The results in SPD demonstrated (1) reduced thrombus dimensions in delta-SPD (pure dense granule deficiency) in proportion to the magnitude of the dense granule defect; (2) an even greater reduction in thrombus dimensions in patients with combined deficiencies of alpha and dense granules (alpha delta-SPD); and (3) impaired platelet adhesion at several conditions in alpha delta-SPD and, in delta-SPD, a hematocrit-dependent impairment of adhesion in citrated blood at 2,600 sec-1. In thrombasthenia, platelets were present as a monolayer on the subendothelial surface in both nonanticoagulated and citrated blood, indicating an absolute requirement for GPIIb-IIIa in promoting platelet-platelet interaction at all shear rates and perfusion times. Two types of abnormalities in platelet-vessel wall interactions were observed. In nonanticoagulated blood, the percentage of platelets in the C phase was consistently increased at all shear rates, but C + S values were normal. These observations indicate that platelets deficient in GPIIb-IIIa do not spread normally on the subendothelial surface exposed to nonanticoagulated blood. With citrated blood, the C + S value in thrombasthenia was reduced at both 800 and 2,600 sec-1, as in von Willebrand's disease, and a similar degree of reduction (about 50%) was observed in normal blood treated with a monoclonal antibody to GPIIb- IIIa. The findings, together with theoretical considerations, are consistent with an hypothesis that GPIIb-IIIa mediates the spreading of platelets on subendothelium following the initial attachment through GPIb and that GPIIb-IIIa may be considered an adhesion site on the platelet membrane. Abnormalities of GPIIb-IIIa may, depending on the conditions of study, result in either increased values of C platelets or decreased values of C + S. The results of the study further suggest that a complex interaction of platelet granule factors and membrane GP mediate platelet adhesion and thrombus formation.     

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.     

Selective reduction of serotonin storage and ATP release in chronic renal failure patients platelets

Hemorrhagic complications, as monitored by skin bleeding times, occur in a significant number of chronic renal failure (CRF) patients. The etiology of hemostatic defects in these patients is complex and ill defined. Our studies demonstrate, for the first time, that activated platelets, derived from CRF patients, release significantly (P less than .001) less ATP than controls while the percent of releasable serotonin (5HT), assumed to be co-stored with ATP, is unaltered. Analysis of the CRF-derived platelets reflects a selective acquired storage pool defect with significantly (P less than .001) reduced 5HT levels while their dense granule contents of ATP and ADP are normal. The comparison of ATP release from platelets derived from CRF patients whose bleeding times were less than 9 min to those with bleeding times of 9 min or greater was significantly different (P less than .02). This report demonstrates for the first time that there is a statistically significant correlation of ATP release and 5HT content to bleeding times (P less than .001). The perturbation of platelet 5HT uptake, 5HT dense granule content, and ATP release appears to result from newly described altered plasma factors, detected by our in vitro mixing studies. It is proposed that the reduced level of releasable platelet 5HT and ATP contributes to bleeding disorders commonly encountered in CRF patients.     

Platelet density analysis: a tool for the detection of acquired storage pool disease in man

S ummary . This study was designed to evaluate the usefulness of platelet density analysis in the detection of acquired storage pool defects in human patients. Two groups of patients were investigated: 19 subjects affected with a myeloproliferative disorder (group I) where abnormal platelets are released from the megakaryocytes and 11 patients hospitalized in an intensive care unit (Group II) where normal platelets are injured in the circulation. Platelet density distribution after isopycnic centrifugation on a discontinuous stractan density gradient, dense granule markers (serotonin, ATP and ADP) and alpha granule markers (intraplatelet beta-thromboglobulin and platelet factor 4) were simultaneously determined. An increased proportion of the percentage of light platelets was observed in 16 patients of group I and nine of group II; an increased ATP/ADP ratio was observed in 12 patients of group I and 10 of group II. Both the tests were abnormal in 11 patients of group I and nine of group II. In group I, the level of serotonin was low and was related to the percentage of light platelets. The alpha granule specific proteins were normal in the two groups. These results indicate that platelet density analysis may serve as a screening test to detect exhausted platelets in human diseases.     

The secretory release reaction initiated by complement proteins C5b-9 occurs without platelet aggregation through glycoprotein IIb-IIIa

The secretory and aggregation responses of stirred platelets exposed to complement proteins C5b-9 was investigated. C5b-9 assembly on the platelet surface resulted in the release of dense granule adenosine triphosphate (ATP) accompanied by a decrease in sample turbidity, but no detectable cell lysis. Inhibition of cellular protein kinase C completely blocked the C5b-9 initiated release of ATP, confirming the secretory nature of this response. Addition of fibrinogen (up to 1 mg/mL) had no effect on either the release of ATP or the decreased turbidity observed for C5b-9 cells. Addition of the peptides Arg-Gly- Asp-Ser (RGDS) and fibrinogen gamma-chain carboxyl-terminal (gamma 397- 411) at concentrations sufficient to completely block fibrinogen binding to GP IIb-IIIa had no effect on either C5b-9 induced dense granule secretion or the associated turbidity change. Microscopic examination and electronic particle counting of the stirred platelet suspensions confirmed that no aggregation of C5b-9 platelets occurred even when these cells were stirred in the presence of fibrinogen. The capacity of the C5b-9 proteins to initiate platelet secretion without activation of cell surface glycoprotein (GP) IIb-IIIa suggests a mechanism for intravascular dissemination of activated platelets during complement activation in vivo.     

Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes

The labelling of platelets with thiazole orange (TO) has been utilized by various laboratories to determine the percentage of reticulated platelets within whole blood or platelet-rich plasma (PRP). A proportion of TO labelling, however, is not entirely mRNA specific and remains to be fully defined. Almost half of the total TO-positive signal within normal platelets ( n  = 5) was shown to be abrogated upon degranulation with 80 μ m thrombin receptor activating peptide (TRAP) ( P  = 0.006), strongly suggesting that platelet granules are non-specifically labelling with dye. We have confirmed this hypothesis by studying TO labelling of platelets within whole blood from dense granule deficient patients, e.g. Hermansky-Pudlak syndrome (HPS) ( n  = 5) and storage pool disease (SPD) ( n  = 4). The levels of TO-positive platelets were found to be significantly lower than normal ( P  = 0.0003 and P  = 0.0002 respectively), but not significantly different from TRAP degranulated platelets. Upon degranulation of HPS and SPD platelets there was very little further reduction in the TO signal. Incubation of normals and SPD whole blood with different concentrations of either TO or coriphosphine-O confirmed that dense granules were non-specifically labelling even at high concentrations of both dyes. These findings suggest that although TO labelling is in part RNA specific, the dense granular pool of nucleotides appears to cause a substantial amount (∼50%) of non-specific labelling observed under these conditions of assay. This can easily be controlled for by a degranulation step with a non-enzymatic platelet agonist such as TRAP, and may have important consequences for the eventual standardization, clinical utilization and automation of reticulated platelet assays.     

Initial characterization of human platelet mepacrine-labelled granules isolated using a short metrizamide gradient

S ummary . A method for the purification of human platelet mepacrine-labelled granules is described. Characterization of these isolated granules allowed them to be identified as the serotonin storage organelles or dense bodies. Each step of the purification procedure has been controlled in order to obtain a minimum of leakage of the granule content during initial isolation of the platelets from the blood, the platelet washing procedures, and platelet lysis and the subcellular separation. A key step in the procedure was the centrifugation of the labelled granules across a short, discontinuous metrizamide gradient. The pellet of isolated mepacrine-fluorescent granules consisted almost entirely of granules with the typical appearance of dense bodies, as shown by electron microscopy, and was relatively free from membranes and other granule populations as evaluated by the presence of the different markers (tritiated lectin, beta-glucuronidase, monoamine oxidase, platelet factor 4). The method is simple, reproducible and allows the highest enrichment in dense bodies obtained hitherto with human platelets: x 177 in calcium and x 115 in [¹⁴C]serotonin after fractionation of [¹⁴C]serotonin-labelled whole platelets. Functional studies performed with the isolated granules showed that they rapidly accumulated [¹⁴C]serotonin.     

Unproportionally high concentrations of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in heavy platelets

Summary Platelets from whole blood were separated into five density subpopulations using a discontinuous Percoll gradient. The content of diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), ADP and ATP were determined in the subfractions. The dinucleotides were directly measured in neutralized, acid-soluble extracts of human platelets with a bioluminescence method not requiring any chromatographic step. When comparing the nucleotide contents of the density subpopulations it became evident that all nucleotides steadily increased with increasing density. Ap₃A, Ap₄A, ADP and ATP were present in 10-, 7-, 4-and 2-fold higher amounts in the heaviest platelets, respectively, as compared to the subfraction with the lowest density. This finding is practically relevant since the most dense platelet subpopulations may be lost during conventional centrifugation to obtain platelet-rich plasma. Therefore we compared a platelet population obtained from PRP with the platelet population, which had been prepared from whole blood by means of a continuous Percoll gradient. All the four nucleotides investigated were represented in 1.5- to 2-fold higher amounts in the whole blood platelet population. This indicates that PRP does not contain a representative population but lacks part of the large heavy platelets containing the highest amounts of nucleotides.Abbreviations ACD (citric) acid-citrate-dextrose - AMR ATP Monitoring Reagent^® (LKB) - Ap₃A diadenosine triphosphate - Ap₄A diadenosine tetraphosphate - BSCG buffered saline-glucose-citrate - PDE phosphodiesterase - PEP phosphoenolpyruvate - PK pyruvate kinase - PRP platelet-rich plasma - TCA trichloroacetic acid - Tris Tris-(hydroxymethyl)aminomethane This work is dedicated to Prof. Dr. Walter Kersten on the occasion of his 60th birthday     

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.     

Hydrophobic organic solvents activate human platelets in vitro

The purpose of this study was to investigate blood platelet function during exposure to the hydrophobic organic solvents toluene, p-xylene and n-hexane. Human blood platelets were exposed for 30 min at 37°C to a saturated atmosphere of p-xylene, toluene or n-hexane. All three solvents, and the aromatics in particular, induced a decrease in the number of single platelets (61-88%) together with an increase in the extracellular levels of ATP plus ADP (45-65% of total) and serotonin (67-100% of total). Passive leakage of [(14)C] adenine-labelled nucleotides from the metabolic pool, due to platelet lysis, was minor or delayed. Electron microscopy of platelets exposed to p-xylene revealed aggregation. The platelets were spherical without pseudopods. Our results indicate that the hydrophobic solvents n-hexane, p-xylene and toluene induce platelet aggregation and dense granule secretion.     

Immature dense granules in platelets from mice with platelet 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.     

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.     

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.