Weak correlation of bleeding scores to platelet electron microscopy: A retrospective chart review of pediatric patients with delta-storage pool disorder.

BACKGROUND: Delta granule storage pool deficiency (δ-SPD) is a rare platelet disorder in which a deficiency of platelet granules leads to poor aggregation, resulting in varying clinical bleeding phenotypes. Children with δ-SPD have variable laboratory results, making the proper diagnosis and evaluation controversial. OBJECTIVES: To describe the demographic and laboratory trends of this population and to assess the value of electron microscopy in diagnostic evaluation and its correlation to bleeding symptoms. METHODS: We performed a retrospective review of 109 pediatric patients diagnosed with δ-SPD. We collected demographic information and bleeding scores using a validated bleeding assessment tool. A descriptive and exploratory analysis was performed. RESULTS: The majority of patients were female, with an average age at diagnosis of 11.61 years. Females were diagnosed at a significantly older age presenting most often with menorrhagia, while males presented most commonly with epistaxis. The majority showed normal lumiaggregometry, the mean platelet electron microscopy (PEM) value was 2.37, and the mean bleeding score was 6. Bleeding assessment tool and PEM had a significantly weak correlation. CONCLUSIONS: Patients with more dense granules per platelet had higher bleeding scores than those with fewer dense granules per platelet. The current body of evidence does not favor the use of PEM in routine clinical practice, and results are difficult to interpret. In patients with severe mucocutaneous bleeding symptoms and normal platelet aggregation studies, consideration should be given to an alternative diagnosis and further evaluation is warranted.

Angiomatoid Fibrous Histiocytoma With Paraneoplastic Platelet Storage Pool Deficiency.

Angiomatoid fibrous histiocytoma is a rare soft tissue tumor usually discovered in young individuals. This tumor is often mistaken for a hematoma and typically misdiagnosed. It is commonly found in the extremities and may be associated with a site of recent or previous trauma. Characteristic histology includes nodules of histiocytoid spindle cells with pseudoangiomatoid spaces, fibrous pseudocapsules, and lymphoid cuffing. We describe the case of an 8-year-old girl who presented after incision and drainage of a superficial thigh lesion and experienced subsequent chronic bleeding of her wound. Her initial presentation was concerning for an underlying bleeding disorder, and laboratory analysis uncovered a paraneoplastic platelet function disorder that resolved with therapy of the primary tumor.

Storage pool diseases illuminate platelet dense granule biogenesis.

Platelet dense granules (DGs) are membrane bound compartments that store polyphosphate and small molecules such as ADP, ATP, Ca2+, and serotonin. The release of DG contents plays a central role in platelet aggregation to form a hemostatic plug. Accordingly, congenital deficiencies in the biogenesis of platelet DGs underlie human genetic disorders that cause storage pool disease and manifest with prolonged bleeding. DGs belong to a family of lysosome-related organelles, which also includes melanosomes, the compartments where the melanin pigments are synthesized. These organelles share several characteristics including an acidic lumen and, at least in part, the molecular machinery involved in their biogenesis. As a result, many genes affect both DG and melanosome biogenesis and the corresponding patients present not only with bleeding but also with oculocutaneous albinism. The identification and characterization of such genes has been instrumental in dissecting the pathways responsible for organelle biogenesis. Because the study of melanosome biogenesis has advanced more rapidly, this knowledge has been extrapolated to explain how DGs are produced. However, some progress has recently been made in studying platelet DG biogenesis directly in megakaryocytes and megakaryocytoid cells. DGs originate from an endosomal intermediate compartment, the multivesicular body. Maturation and differentiation into a DG begins when newly synthesized DG-specific proteins are delivered from early/recycling endosomal compartments. The machinery that orchestrates this vesicular trafficking is composed of a combination of both ubiquitous and cell type-specific proteins. Here, we review the current knowledge on DG biogenesis. In particular, we focus on the individual human and murine genes encoding the molecular machinery involved in this process and how their deficiencies result in disease.

Procoagulant platelets: are they necrotic?

Apoptosis and necrosis represent distinct cell death processes that regulate mammalian development, physiology and disease. Apoptosis characteristically leads to the silent destruction and removal of cells in the absence of an inflammatory response. In contrast, necrotic cell death can induce physiologic inflammatory responses linked to tissue defense and repair. Although anucleate, platelets undergo programmed cell death, with apoptosis playing an important role in clearing effete platelets from the circulation. While it has long been recognized that procoagulant platelets exhibit characteristic features of dying cells, recent studies have demonstrated that platelet procoagulant function can occur independent of apoptosis. A growing body of evidence suggest that the biochemical, morphologic and functional changes underlying agonist-induced platelet procoagulant function are broadly consistent with cell necrosis, raising the possibility that distinct death pathways regulate platelet function and survival. In this article, we will discuss the mechanisms underlying apoptotic and necrotic cell death pathways and examine the evidence linking these pathways to the platelet procoagulant response. We will also discuss the potential contribution of these pathways to the platelet storage lesion and propose a simplified nomenclature to describe procoagulant platelets.

Platelet structural pathology in a patient with the X-linked GATA-1, R216Q mutation.

The ultrastructural pathology of GATA-1, V205M and G208S macrothrombocytes was discussed in earlier investigations. This study has used the same technology to evaluate macrothrombocytes from a patient with the GATA-1, R216Q mutation. Some of the pathological features observed in macrothrombocytes from patients with the V205M and G208S variations including hypo- and agranular platelets, tubular inclusions and platelets within platelets, as well as platelets within platelets within platelets were identified. However, tubular membrane sheets in megakaryocytes and platelets of the V205M and G208S types and large groups of platelets attached to platelets to form megathrombocytes were not observed. The unique pathology of the megathrombocytes from this patient was the near absence of dense bodies in his giant cells. Storage Pool Deficiency, together with large platelets, defective adhesion and aggregation of his macrocytes under shear stress to vWF and collagen and defective clot retraction may contribute to the pathogenesis of his bleeding disorder.

The role of Rab38 in platelet dense granule defects.

BACKGROUND: The fawn-hooded hypertensive (FHH) rat has a mutation in the Rab38 gene that is associated with a platelet dense granule storage pool disease. OBJECTIVE: To better characterize the expression and function of Rab38 in FHH rat and human megakaryocytes and platelets. PATIENTS AND METHODS: Rab38 expression in FHH rat and normal tissues was demonstrated by western blotting. Platelet and megakaryocyte morphology and Rab38 expression were examined by transmission electron microscopy and by immunofluorescence confocal microscopy. Platelet surface glycoprotein and P-selectin expression and total serotonin content were assessed by flow cytometry. RESULTS: Rab38 was not expressed in FHH rat tissues, and FHH rat platelets and megakaryocytes lacked dense granules. FHH rat platelets had normal expression of surface glycoproteins and of surface P-selectin in response to thrombin. The total serotonin content in FHH rat platelets was similar to that in Brown Norway rat platelets. In a megakaryocyte cell line, Rab38 was expressed in a granular perinuclear and cytoplasmic pattern. There was partial colocalization with serotonin, and minimal colocalization with von Willebrand factor and lysosomal proteins. CONCLUSIONS: The lack of Rab38 expression in the FHH rat results in the absence of normal dense granules in the megakaryocytes and platelets, which have otherwise normal structure and function. Rab38 may play a role in the development of dense granules in the megakaryocytes and platelets.

A clinical variant of familial Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive inherited disease consisting of (1) partial oculocutaneous albinism (with nystagmus, strabism, and visual acuity loss), (2) platelet storage pool deficiency (with bleeding diathesis), and (3) disorder of "ceroid" metabolism with a multisystem tissue lysosomal ceroid deposition. HPS is less uncommon in Puerto Rico, where the most important studies have been performed, but is a very rare disease in Europe. HPS basic defect remains unknown, even if an HPS-causing gene was identified in chromosome segment 10q23-q23.3, and several mutations have been reported. The aim of this article is to discuss, on the basis of a review of relevant literature, a new familial HPS clinical variant observed in 2 young sisters (aged 16 and 23 years old, respectively), characterized by the typical symptoms of this syndrome. Our patients also suffered from diffuse interstitial pulmonary disease and an unexpectedly increased platelet aggregation and were prone to bacterial infections. Interestingly, we observed urinary tract abnormality in the younger HPS sister and a porencephalic cyst in the older HPS sister; both of these developmental defects have been reported in the Cross syndrome (or oculocerebral hypopigmentation syndrome). It seems that in our patients, an overlapping of the phenotypic manifestations of different rare syndromes may be present. The presence of ceroid-like autofluorescent material in urinary sediment together with the histologic aspects and the autofluorescence of oral mucosa biopsy are consistent with a ceroid-like lipofuscin storage. HPS should be carefully tested for in suspected cases to prevent the severe visual impairment, rapidly progressive pulmonary fibrosis, and other complications associated with this disorder.

Acquired storage-pool disorders occurring late after allogeneic bone marrow transplantation: partial activation of platelets in asymptomatic patients.

Bone marrow transplantation (BMT) may be complicated by coagulation abnormalities. The present study evaluated whether platelets might be activated in patients who had undergone BMT without significant coagulopathy. The patients selected had received allogeneic BMTs a median of 39 months before the study (range, 11-124 months) and had not received cyclosporine, FK506 (tacrolimus), or other medication affecting cyclo-oxygenase for at least 3 months prior to the collection of blood samples. Furthermore, patients had platelet counts greater than 100 x 10(9) cells/L and normal serum creatinine levels. Twenty-five healthy volunteers acted as controls. Platelet aggregation studies and a mepacrine assay of platelets showed abnormal aggregation and decreased staining in some patients. The platelet storage-pool adenosine 5'-triphosphate (ATP) level in 15 patients after BMT was 0.45+/-0.24 micromol per 10(11) platelets, whereas the level in 18 controls was 1.03+/-0.36 micromol per 10(11) platelets (P = .00078). The total ATP levels of platelets in patients and controls were 4.33+/-1.14 and 5.63+/-1.51 micromol per 10(11) platelets, respectively (P = .016). With the exception of 1 patient, plasma levels of thrombomodulin and von Willebrand factor were all within the normal range. The average plasma level of 11-dehydrothromboxane B2 was significantly increased in 15 patients after BMT compared with controls, 20.6+/-8.2 and 10.3+/-1.2 pg/mL, respectively (P = .0004). These findings suggest a long-term process of platelet activation in patients after BMT and, following the cessation of cyclosporine, development of acquired storage-pool disorder of platelets.

Hermansky-Pudlak syndrome: models for intracellular vesicle formation.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by pigment dilution, nystagmus, decreased visual acuity, a bleeding diathesis, and lysosomal accumulation of ceroid lipofuscin. Electron microscopic evidence demonstrating lack of platelet-dense bodies provides the sine qua non for diagnosing HPS. Ceroid lipofuscinosis is considered to cause several serious complications, including progressive pulmonary fibrosis leading to death in the fourth or fifth decades. Currently, only symptomatic treatment can be offered. Although rare in the general population, HPS occurs in northwest Puerto Rico with a prevalence of 1 in 1800. HPS1, the first gene found to be responsible for HPS, was mapped to chromosome 10q23 and subsequently isolated and sequenced. It consists of 20 exons encoding a 700-amino acid, 79.3-kDa peptide with no homology to any known protein. All 10 HPS1 mutations reported to date, including the 16-bp duplication found in all northwest Puerto Rican patients, result in truncated proteins. The two mutations in the mouse pale ear gene (ep), which is the murine homology of HPS1, cause similarly truncated proteins. The pathologic nature of these truncation mutations may result from unstable mRNA. However, in combination with the absence of any disease-causing missense mutations, it may indicate that the C-terminus of the HPS1 peptide is functionally important. The disorder HPS displays locus heterogeneity, consistent with the existence of 14 mouse strains manifesting both hypopigmentation and a platelet storage pool deficiency. Two mouse models, pearl and mocha, have mutations in the beta3A and delta subunits of the adaptor-3 complex, respectively. This suggests that defective vesicular trafficking, specifically cargo packaging, vesicle formation, vesicle docking, or membrane fusion, may comprise the basic defect in HPS. Studies of the proteins involved in intercompartmental transport for melanosomes, platelet-dense bodies, and lysosomes should lead to a better understanding of the mechanisms of organellogenesis and to more effective therapies for HPS.

[Thrombocyte dysfunction in children with albinism]. / Thrombocytafunkció-zavar albínó gyermekekben.

Seven children with albinism were examined for haemorrhagic diathesis. In 6 patients coagulation screening tests (prothrombin time, activated partial thromboplastin time, thrombin time) were normal, in 1 patient prolongation of activated partial thromboplastin time due to mild factor XI deficiency was found. Platelet counts were in the reference range, however, the template bleeding time of 6 out of 7 patients was prolonged. Evaluation of platelet aggregation and secretion in a lumi-aggregometer demonstrated that only the child with normal bleeding time had normal platelet function, and 6 children with prolonged bleeding time had impaired aggregation and release reaction. Five out of these 6 patients had severe decrease of ATP secretion even when high dose of thrombin was used as agonist. The number of dens granules in the platelets of these patients, as measured by the uptake of mepacrine, was significantly less than in normal controls. These findings clearly suggested storage pool deficiency. In one of these patients storage pool deficiency was associated with mild factor XI deficiency. The high frequency of haemorrhagic diathesis in albino children emphasizes, the importance of the screening of patients with albinism for bleeding diathesis.

Flow-cytometric analysis of mepacrine-labelled platelets in patients with end-stage renal failure.

Platelet dysfunction and increased bleeding tendency has been the most consistently described haemostatic abnormality in patients with renal failure. Besides abnormalities in platelet membrane glycoproteins, a reduced amount of platelet-dense granule content has been demonstrated in patients with end-stage renal failure (ESRD) indicating an acquired storage pool deficiency (SPD) present in uraemia. To study dense granules, platelets were labelled with mepacrine, a fluorescent probe which is specifically incorporated into dense bodies. MepaPlatelets of 13 patients with ESRD and of 11 healthy controls were studied. The results showed that mepacrine-labelled platelets of patients with ESRD reveal a significantly (p < 0.05) reduced fluorescence compared to the control group. This implies a reduced number or content of dense granules present in ESRD platelets. Thus, the current data indicate that ESRD is associated with an acquired platelet SPD which may be a useful and rapid method for screening patients with suspected acquired or inherited SPD.

[Acquired platelet storage pool deficiency in rheumatoid arthritis]. / Nabyta choroba puli magazynowej plytek krwi w przebiegu reumatoidalnego zapalenia stawów.

A 64-year-old woman with a 15-years-history of rheumatoid arthritis developed generalized hemorrhagic diathesis. Routine coagulation tests revealed a slightly diminished platelet count only. Platelet aggregation in vitro induced by ADP, collagen, thrombin, arachidonic acid and ristocetin were reduced. The patient's plasma aggregating activity was significantly diminished which was due to a decrease of the intraplatelet nucleotide pool. The number of mepacrine labelled bodies as well as dense bodies in electron microscopy was below the normal values as well. Moreover, the intraplatelet concentration of cyclooxygenase--malonylodialdehyde (MDA) and lipoxygenase pathway products were lowered. Total platelet immunoglobulin G and M contents were significantly increased. The platelet survival time (in vitro aspirin method) was slightly shortened. Finally the diagnosis of delta-acquired platelet storage pool deficiency (delta-SPD) was established and possibilities of treatment were discussed.

Hermansky-Pudlak syndrome (HPS). An epidemiologic study.

A study of albinism in Puerto Rico identified 693 persons with albinism. Among these, the type of albinism was determined in 595, Hermansky-Pudlak syndrome (HPS) was found in 495. Approximately five of every six Puerto Rican albinos had HPS. The highest prevalence of HPS yet reported was in the northwestern quarter of the island where at least 1 in 1,800 persons had HPS, and approximately 1 in 21 were carriers. The HPS albino pigment phenotype was variable, and HPS albinos phenotypically resembled other types of oculocutaneous and ocular albinos. Ceroid storage was also variable. The consistent finding in HPS was storage pool deficient platelets. HPS is best diagnosed by lack of platelet dense bodies seen by electron microscopy. Evidence from family studies indicates that HPS is a distinct disorder due to the pleiotropic effects of a single gene mutation or a small deletion.

Compartmentation of 4,6-difluoro-5HT studied by nuclear magnetic resonance in normal and CHS bovine platelets.

A platelet storage pool deficiency (SPD) is present in platelets from cattle with the Chediak-Higashi syndrome (CHS). The most plausible hypothesis for the SPD is that dense granule precursors are simply not formed in CHS megakaryocytes. There is, however, evidence that some recently acquired 5-hydroxytryptamine (5HT) is located in granules and that the granules have an acidic interior. To obtain a greater understanding of the processing of 5HT by SPD platelets, normal and CHS platelets were incubated with 4,6-difluoro-5HT and studied by 19F NMR at 188 mHz. Normal platelets contained 2 compartments for 4,6-difluoro-5HT as indicated by 2 well-developed resonances for each 19F. The resonances were unequal in magnitude. The predominant resonance broadened with lower temperatures and was absent in CHS bovine platelets; it was, therefore, the dense granule compartment. There was only 1 resonance for each 19F in CHS platelets. The chemical shift was identical to the minor resonance, or non-dense granule resonance, found in normal bovine platelets but the resonance width was increased, indicating that some non-dense granule 4,6-difluoro-5HT was in a more restricted environment within CHS platelets than it was in normal platelets.

[The induction of an acquired storage pool deficiency of the thrombocytes by IgG antibodies in systemic lupus erythematosus and chronic polyarthritis]. / Induktion einer 'acquired storage pool deficiency' der Thrombozyten durch IgG-Autoantikörper bei systemischem Lupus erythematodes und chronischer Polyarthritis.

Sera of patients with RA and SLE contain platelet-reactive IgG antibodies, which bring about the release of serotonin from platelets. They can also induce the aggregation of normal human platelets. In addition, such antibodies cause a decreased in vitro disaggregation of platelets after ADP exposure, as well as a significantly reduced collagen-induced aggregation. Thus, IgG platelet antibodies in RA and SLE can cause a functional impairment of patients' platelets, and may also be instrumental in systemic inflammatory rheumatoid disease by releasing proinflammatory mediators.

[Megaloblastic anemia and platelet function--a qualitative platelet defect in pernicious anemia].

In order to know the entities of platelet defect in pernicious anemia, we investigated platelet functions in ten cases with pernicious anemia. Three of the cases had total gastrectomy. Five cases showed thrombocytopenia and four cases revealed prolongation of Ivy bleeding time. Decreased adhesiveness was observed in three cases. Various abnormalities in platelet aggregation were observed. However, almost all of the cases showed remarkable improvement of decreased platelet functions after the therapy of Vitamin B12 injection. The results of adenine nucleotides in platelets and the release of them following collagen or epinephrine aggregation were analysed in comparison with normal platelets. The ADP was definitely decreased and the ATP/ADP ratio was increased. In addition, the release of ATP and ADP at collagen or epinephrine induced aggregation was markedly decreased, and after the therapy of Vitamin B12, the decrease of adenine nucleotide release remarkably increased. In summary, the acquired defects of platelet function in pernicious anemia are regarded as a secondary storage pool disease, and its defects improve after Vitamin B12 therapy.

[Examination of platelet aggregation and ultrastructure in patients with non-Hodgkin's lymphoma]. / Untersuchungen der Thrombozytenaggregation und Ultrastruktur bei Patienten mit NHL.

Aggregation and secretion reaction of thrombocytes were studied in 54 patients suffering from Non-Hodgkin's++Lymphoma. Results were compared with electron-microscopic findings on thrombocytes of the same patients. Slight changes were found in stage II. In stage IV distinct therapy-resistant changes were noted in combination with storage pool disease syndrome which is to be considered as a consequence of impaired generation of thrombocytes in bone marrow.

Acquired von Willebrand disease and storage pool disease in chronic myelocytic leukemia.

Platelet function was evaluated in 20 patients with chronic myelocytic leukemia (CML), all Ph positive. Seven showed abnormal epinephrine-induced aggregation, while four had impaired both ADP- and collagen-induced aggregation. The platelets of all patients aggregated with arachidonic acid, thus ruling out cyclooxygenase or lipoxygenase deficiency. The intracellular concentrations of ATP and ADP were significantly below normal, and the ratio of ATP/ADP was greater than normal in all 12 patients. ATP released from platelets by Lumi-aggregometer was reduced. In four patients with abnormal ristocetin-induced aggregation, vWF:Ag, RCoF, and FVIII:C were all reduced. No significant inactivation of factor VIII was induced in normal plasma by incubation with patient's plasma. The crossed immunoelectrophoretic analysis revealed that vWF:Ag in these patients was mainly composed of more anodic component as compared with that of normal plasma. The ratio of vWF:Ag/RCoF was significantly greater than normal. A marked increase of factor VIII and a rapid return of vWF:Ag and RCoF to the baseline after the 1-deamino-8-arginine vasopressin (DDAVP) infusion were observed. Transient increase in vWF:Ag after the infusion of DDAVP appeared with less anodic forms and in the same relative proportion as that in normal plasma. The present study shows that in some patients with CML storage pool disease occurs with acquired von Willebrand disease.