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.     

Platelet storage pool deficiency in pigs

We report a new bleeding disease--storage pool deficiency (SPD) of platelets--in pigs from the Mayo swine colony of homozygous von Willebrand's disease (vWD) and of heterozygous carriers of vWD. Levels of factor VIII, von Willebrand factor antigen (vWF:Ag), and ristocetin cofactor (RCof) were similar in the vWD carriers and SPD pigs. The latter pigs, however, had bleeding times of 15 minutes or more and were severe bleeders, in contrast to clinically normal vWD carriers. Platelet aggregation in response to collagen was reduced in most SPD pigs. Total platelet content of ADP, ATP, and serotonin was less than that of normal pigs. While the initial uptake of 14C-labeled serotonin into platelets was similar in SPD and normal pigs, retention of serotonin was reduced in platelets of SPD pigs. Transmission electron microscopy showed a large decrease of dense bodies in the platelets of SPD pigs. These findings support a diagnosis of SPD. Genetic analyses suggest an autosomal recessive mode of inheritance. A breeding program is under way to produce pigs affected only at the SPD gene, thus allowing further characterization of SPD and SPD-carrier pigs.     

Platelet storage pool deficiency in Jacobsen syndrome

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.     

Platelet storage pool deficiency in Jacobsen syndrome

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.     

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.     

Platelet storage pool deficiency: diagnosis in patients with prolonged bleeding times and normal platelet aggregation

We evaluated 46 patients with prolonged bleeding times, and no demonstrable abnormalities of either von Willebrand factor or platelet aggregation, for possible deficiency of platelet storage pool. Studies of ATP release from thrombin-stimulated platelets and enumeration of dense granules in platelet whole mounts were performed in these patients. Seventeen patients (35%) had both decreased ATP release and decreased numbers of dense granules, suggesting the presence of a storage pool defect. Thus, storage pool deficiency may be present in the absence of the classical aggregation abnormalities. Evidence of storage pool deficiency should be considered in all patients with an isolated unexplained prolongation of the bleeding time. The methods used in this study are readily applicable to most clinical laboratories.     

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 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.     

Alpha-delta platelet storage pool deficiency in three generations

Alpha-Delta platelet storage pool deficiency (alphadelta SPD) is a rare inherited bleeding disorder affecting both males and females, occurring in families, as well as sporadically. Patient platelets in most cases are moderately deficient in both alpha granules and dense bodies. Only one patient has been severely deficient in both organelles. The present study is the first to document a severe decrease in both platelet alpha granules and dense bodies in four members in three generations of the same family. Efforts to differentiate this disorder from other hypogranular platelets syndromes in the present investigation suggested that the alpha granules and dense bodies become connected to channels of the open canalicular system (OCS) and lose their contents to the exterior without prior activation of the cells. In contrast, alpha granule formation in the white platelet syndrome is too slow, and cells leave the bone marrow still in the process of producing organelles. Gray platelet syndrome platelets can make alpha granules, but their enclosing membranes are unable to retain stored products. As a result, the organelles lose their contents to surrounding cytoplasm in megakaryocytes and platelets, not selectively through the demarcation system channels and OCS channels. Thus, the pathogenesis of alphadelta SPD is unique.     

Novel mutations associated with combined pituitary hormone deficiency

The pituitary gland produces hormones that play important roles in both the development and the homeostasis of the body. A deficiency of two or several of these pituitary hormones, known as combined pituitary hormone deficiency, may present in infants or children due to an unknown etiology and is considered congenital or idiopathic. Advancements in our understanding of pituitary development have provided a genetic basis to explain the pathophysiological basis of pituitary hormone disease. Nevertheless, there are several challenges to the precise characterization of abnormal genotypes; these exist secondary to the complexities of several of the hypothalamic/pituitary developmental factors and signals, which ultimately integrate in a temporal and spatial dependent manner to produce a mature gland. Furthermore, the clinical presentation of pituitary hormone disease may be dynamic as subsequent hormone deficiencies may develop over time. The characterization of patients with mutations in genes responsible for pituitary development provides an opportunity to discover potential novel mechanisms responsible for pituitary pathophysiology. The focus of this review is to report the most recent mutations in genes responsible for pituitary development in patients with hypopituitarism and emphasize the importance to physicians and researchers for characterizing these patients. Continuing efforts toward understanding the molecular basis of pituitary development as well as genetic screening of patients with pituitary disease will offer new insights into both diagnostic and potential therapeutic options that will decrease the morbidity and mortality in patients with hypopituitarism.     

Platelet malondialdehyde production and aggregation responses induced by arachidonate, prostaglandin-G2, collagen, and epinephrine in 12 patients with storage pool deficiency

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta- SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.     

Progenitor cell defect correctable by bone marrow transplantation in five independent mouse models of platelet storage pool deficiency

Two human platelet storage pool deficiencies (SPD), Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited and characterized by hypopigmentation, prolonged bleeding, and normal platelet numbers accompanied by a reduction of platelet dense granules. Seven independent and unique mouse pigment mutations regulated by separate genes have been proposed as animal models for SPD. Mice homozygous for the recessive mutations have diluted pigmentation, prolonged bleeding times, normal platelet concentrations, and reduced numbers of platelet dense granules. Reciprocal bone marrow transplantations were carried out between normal C57Bl/6J mice and five of these mutants, pearl, light ear, pale ear, ruby-eye, and maroon, to test whether the platelet defects are due to platelet progenitor cells or to humoral regulatory factors. Recipient mice were transplanted with marrow after 950-rad whole body irradiation. The prolonged bleeding time and low serotonin concentrations of the five mutants were converted to normal values after transplantation with normal marrow. Normal mice displayed characteristics of platelet SPD when transplanted with mutant marrow. This study demonstrates that in each of five independent mouse models the thrombopathy of SPD is due to a platelet progenitor cell defect correctable by bone marrow transplantation. These findings suggest that in severe cases human SPD may be amenable to treatment by bone marrow transplantation.     

Heterozygous factor XI deficiency associated with three novel mutations

To determine the utility of single-stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC --> TGC (Arg308Cys) mutation in exon 9, a GCT-->GTT (Ala412Val) mutation in exon 11 and an AGC --> AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.     

Acquired storage pool deficiency with increased platelet-associated IgG. Report of five cases

Acquired abnormalities of platelet aggregation have been reported with increasing frequency. We studied five patients (including two with systemic lupus erythematosus and one with compensated chronic idiopathic thrombocytopenic purpura) in whom platelet aggregation responses to collagen, epinephrine and ADP are impaired; in all cases, we found that levels of platelet-associated immunoglobulin G (IgG) were increased. In all five patients substances stored in platelet-dense granules (ATP, ADP, serotonin and calcium) were diminished. The content of the alpha-granule substance, beta-thromboglobulin, was also decreased in most cases, whereas the levels of two secretable acid hydrolase enzymes (beta-glucuronidase and beta-N-acetyl glucosaminidase) were within normal limits. These findings are similar to those observed in subtypes of congenital storage pool deficiency. However, in contrast to the congenital disorder, a membrane-bound (nonsecretable) acid phosphatase was also decreased in the patients with acquired storage pool deficiency. These findings suggest that impaired platelet aggregation on an acquired basis may, in some patients, be due to immune platelet damage resulting in a distinctive type of platelet storage pool deficiency.     

Identification of genetic loci simultaneously associated with multiple cardiometabolic traits

Background and aimsCardiometabolic disorders (CMD) arise from a constellation of features such as increased adiposity, hyperlipidemia, hypertension and compromised glucose control. Many genetic loci have shown associations with individual CMD-related traits, but no investigations have focused on simultaneously identifying loci showing associations across all domains. We therefore sought to identify loci associated with risk across seven continuous CMD-related traits.Methods and resultsWe conducted separate genome-wide association studies (GWAS) for systolic and diastolic blood pressure (SBP/DBP), hemoglobin A1c (HbA1c), low- and high- density lipoprotein cholesterol (LDL-C/HDL-C), waist-to-hip-ratio (WHR), and triglycerides (TGs) in the UK Biobank (N = 356,574–456,823). Multiple loci reached genome-wide levels of significance (N = 145–333) for each trait, but only four loci (in/near VEGFA, GRB14-COBLL1, KLF14, and RGS19-OPRL1) were associated with risk across all seven traits (P < 5 × 10^?8). We sought replication of these four loci in an independent set of seven trait-specific GWAS meta-analyses. GRB14-COBLL1 showed the most consistent replication, revealing nominally significant associations (P < 0.05) with all traits except DBP.ConclusionsOur analyses suggest that very few loci are associated in the same direction of risk with traits representing the full spectrum of CMD features. We identified four such loci, and an understanding of the pathways between these loci and CMD risk may eventually identify factors that can be used to identify pathologic disturbances that represent broadly beneficial therapeutic targets.     

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.     

Clinical and molecular significance of genetic loci associated with psoriatic arthritis

Psoriatic arthritis (PsA) is caused by a combination of environmental and multiple genetic factors, with clear evidence for a strong genetic basis. The remarkable accumulation of knowledge gained from genetic, pharmacogenetic, and therapeutic response of biologic agents in PsA has fundamentally changed and advanced our understanding of disease pathogenesis and has identified key signalling pathways. However, only one-quarter of the genetic contribution of PsA has been accounted for; and dissecting the genetic contributors of the cutaneous disease from those that would identify joint disease has been challenging. More importantly, the clinical utility of multiple proposed loci is unclear. In this review, we summarize the potential clinical relevance from established genetic associations and provide insight on the proposed molecular pathways that arise from these associations.     

Morphometry of age pigment (lipofuscin) and of ceroid pigment deposits associated with vitamin E deficiency

Consistent amounts of lipofuscin and of ceroid pigment associated with vitamin E deficiency are reported to represent morphological correlates of aging and increased oxidative stress. A reliable quantification of these yellow autofluorescent deposits is of critical biological significance, thus we carried out a computer-assisted morphometric study on the accumulation of lipofuscin in physiological aging and of ceroid pigment in vitamin E deficiency, respectively. The total area and the size distribution of lipofuscin or ceroid pigment deposits were measured in CA3 hippocampal pyramidal neurons of 6-, 12-, 18- and 25-month-old rats, as well as in vitamin E deficient animals of 18 months of age. An increase in the mean total area of lipofuscin and ceroid pigment was found in aging and in vitamin E deficiency. In both conditions, the proportion of large discrete deposits also increased. The similarity of changes observed in old and adult vitamin E deficient animals suggests that the underlying processes initiated by the absence of alpha-tocopherol from the diet of adult rats and in physiological aging may share some common mechanisms.