An Investigation of ABO Blood Type and the Platelet Delta Granule Storage Pool.

Individuals with bleeding tendencies are more likely to have blood type O than blood types A, B, or AB. Platelet storage pool deficiencies are a lesser-known group of bleeding disorders which often go undiagnosed and may account for a significant number of patients with unexplained bleeding defects. We hypothesized that patients with platelet δ-storage pool deficiency might also have a predominance of type O blood. A retrospective review of medical records of 2,020 patients with unexplained bleeding and evaluated for δ-storage pool deficiency was performed. Correlations between dense granule numbers, blood type, and von Willebrand factor were analyzed for statistical differences. 51.5% of blood samples were blood type O compared to an incidence of 44.0% in the U.S. population. There was a significant association of vWF and blood type O but not with the delta storage pool. There is a preponderance of blood type O in the study population compared to the U.S. population. There is no statistically significant link between blood type O and lower dense granule numbers in this study.

Platelet Electron Microscopy: Utilizing LEAN Methodology to Optimize Laboratory Workflow.

BACKGROUND: Platelet electron microscopy (PEM) is the gold standard methodology for diagnosing storage pool disorder, defined as a paucity of delta granules, alpha granules, or both. PEM literature is limited with few published resources and without well-developed interlaboratory standardization for the preparation and examination of platelet samples. METHODS: Whole mount (WM) dense body (DB) counts for 300 pediatric cases were reviewed to determine whether counting fewer platelets could yield the same results. For 6 cases, DB average was determined on the day of WM preparation and on 2 consecutive days. Both WM and thin section (TS) preparations were examined for all cases. RESULTS: Employing LEAN methodology, an algorithm was developed to markedly decrease the number of platelets counted and still ensure accurate results. WMs decay with time; a statistically significant difference in DB counts was noted between day 0 and day 1 (p < .1). Twelve of 300 cases required both WM and TS preparations for a complete diagnosis. CONCLUSION: It is possible to maintain accuracy and decrease 100 platelet DB counts by >75%. WMs must be counted on the day they are prepared to avoid false paucity of DB secondary to sample decay. An accurate evaluation of platelet morphology requires both the WM and TS techniques.

Variation in Platelet Delta Granules Over Time in Young Women Undergoing Evaluation for Heavy Menstrual Bleeding.

Delta-granule platelet storage pool deficiency (δ-PSPD) is a qualitative platelet function defect associated with variable bleeding phenotypes. Platelet electron microscopy (EM) is commonly utilized to evaluate for δ-PSPD, but intrapatient variability in platelet δ-granule numbers by EM is currently unknown. Fifteen young women aged 11 to 17 years presenting to a young women's hematology clinic for the evaluation of heavy menstrual bleeding underwent platelet EM testing at their initial hematology clinic visit and at 1 and 3 months later. Platelet aggregation of platelet-rich plasma by light transmission was also performed on all patients at their initial visit. Eight patients had average δ-granules per platelet consistently ≥2. Three patients were found to have average δ-granules per platelet <2 on initial testing, 2 of which reverted to ≥2 on subsequent testing. When initial average δ-granules per platelet was ≥2, initial repeat testing remained so in 83% (95% confidence interval [CI], 52%-98%) of cases and subsequent repeat testing remained so in 75% (95% CI, 43%-95%) of the cases. Platelet aggregation testing was abnormal in 53% of patients, and there was no apparent correlation between platelet EM findings and platelet aggregation testing. In this small group of young women presenting for the evaluation of bleeding symptoms, we found that almost half of the patients had substantial variability in platelet EM results. Given other identified limitations in platelet EM testing, and the intrapatient variability identified in this study, providers should use caution in utilizing EM in isolation to diagnose δ-PSPD.

Assessment and validation of a defined fluid restriction protocol in the use of subcutaneous desmopressin for children with inherited bleeding disorders.

INTRODUCTION: Despite the availability of subcutaneous desmopressin (1-deamino-8-d-arginine vasopressin, SC-DDAVP) as a haemostatic agent for children with mild bleeding disorders, few publications specifically address the safety or efficacy of this mode of administration. AIM: Our aim was to assess whether a defined fluid restriction protocol was effective in preventing hyponatremia in children receiving perioperative SC-DDAVP, and to document adequate biological and clinical response in this setting. METHODS: We retrospectively analysed a cohort of children with mild bleeding disorders prescribed SC-DDAVP over a 5-year period following institution of a 'two-thirds maintenance' fluid restriction protocol. RESULTS: Sixty-nine patients received SC-DDAVP following this protocol, including 15 with mild haemophilia A, 49 with von Willebrand disease (VWD) and five with platelet storage pool disorder. In patients who underwent formal preoperative assessment a complete or partial response was observed in 28/29 with type 1 VWD and 14/15 with mild haemophilia A. Perioperative SC-DDAVP provided excellent haemostasis in all patients, with no requirement for factor concentrate or blood products. Mild asymptomatic hyponatremia was detected in seven children who received multiple doses of DDAVP (lowest sodium 129 mmol L(-1) ); however, adherence to the prescribed fluid restriction protocol was questionable in six of these cases. Symptomatic hyponatremia was not observed. CONCLUSION: Subcutaneous desmopressin was well-tolerated, with no serious side-effects observed, and good biological responses in preoperative trials. A two-thirds maintenance fluid regimen was effective at preventing symptomatic hyponatremia in our cohort, and is now the standard protocol for fluid restriction post-DDAVP administration in our centre.

Platelets: still a therapeutical target for haemostatic disorders.

Platelets are cytoplasmatic fragments from bone marrow megakaryocytes present in blood. In this work, we review the basis of platelet mechanisms, their participation in syndromes and in arterial thrombosis, and their potential as a target for designing new antithrombotic agents. The option of new biotechnological sources is also explored.

Molecular determinants of platelet delta storage pool deficiencies: an update.

Delta storage pool deficiency (δ-SPD) is a rare heterogeneous group of platelet disorders characterized by a reduction in the number or content of dense granules. δ-SPD causes a mild to moderate bleeding diathesis characterized mainly by mucocutaneous bleeding. Currently, no specific treatment is available and the therapeutic approach is based on prevention of excessive bleeding. However, during the last few years, important insights into the pathophysiology of δ-SPD have been achieved using mouse models and dense granule deficiency-associated congenital diseases, such as Hermansky-Pudlak syndrome and Chediak-Higashi syndrome. It thus appears that δ-SPD represents a genetically heterogeneous group of intracellular vesicle biogenesis and/or trafficking disorders. This review summarizes recent data regarding the molecular mechanisms together with clinical features of the different types of δ-SPD. Although the molecular basis of isolated inherited δ-SPD remains currently unknown, next-generation sequencing strategies should enable researchers to identify the causative genes. Identification of those genes should contribute to our understanding of the pathophysiology, represent useful tools for genetic diagnosis, and eventually lead to new specific therapeutic approaches.

Role of MRP4 (ABCC4) in platelet adenine nucleotide-storage: evidence from patients with delta-storage pool deficiencies.

We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet delta-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet delta-storage pool deficiency (delta-SPD)-like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4 gene did not show any mutation that explained the lack of expression. In platelets with "classic" delta-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other delta-granules membrane marker proteins (eg, LAMP2), was mostly displaced from delta-granules to patches at the plasma membrane, suggesting that platelets with classic delta-SPD have an abnormality that impairs the assembly of normal delta-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new delta-SPD phenotype remains to be elucidated.

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.

Phenotypic heterogeneity in the Gray platelet syndrome extends to the expression of TREM family member, TLT-1.

The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of alpha-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to alpha-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al., Blood 2004; 104: 107-114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.

Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives.

BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

Gray Platelet Syndrome in a Somalian family.

The Gray Platelet Syndrome (GPS) is a rare inherited, hypogranular platelet disorder characterized by virtual absence of alpha granules in bone marrow megakaryocytes and circulating plates. Usually only one member of a family is affected, but families with two or more affected individuals have been reported from France, Australia, and Mexico, and, recently, the United States. The current study has evaluated the first family from East Africa with two affected members, a brother and sister. Neither child has had significant bleeding problems. Electron microscopic studies revealed almost complete absence of alpha granules from their platelets. Instead their platelets were filled with vacuoles similar in size to the missing granules. Dense bodies were normal in number in the girl's platelets, but markedly increased in her brother's cells. Tannic acid staining revealed that very few of the vacuoles were connected to channels of the open canalicular system. As a result, contents of the residual alpha granule vacuoles must leak out of the organelles and diffuse through megakaryocyte and platelet cytoplasm to the outside. The route of escape may differ from other hypogranular platelet syndromes, such as alpha-delta platelet storage pool deficiency.

Electron-dense chains and clusters in platelets from patients with storage pool-deficiency disorders.

Human platelets are known to contain inherently electron-opaque dense bodies, the storage sites for adenine nucleotides and serotonin, which can be identified under the electron microscope without fixation or staining. Recently we have reported that platelets also contain chains and clusters that are also opaque when examined by the whole-mount technique. The possibility that the newly described structures might be stages in the development of dense bodies requires consideration. The present study examined platelets from patients with Hermansky-Pudlak syndrome, Chediak-Higashi syndrome and platelet storage pool disease, whose cells are markedly deficient or lack dense bodies. Platelets from these individuals contained the same frequency of chains and clusters as found in platelets from normal controls. Thus, chains and clusters do not appear related to formation of dense bodies.

Genetic mapping and characterization of the bleeding disorder in the fawn-hooded hypertensive rat.

Release of platelet dense granule contents occurs in response to vascular injury, playing an important role in platelet aggregation and primary hemostasis. Abnormalities of the platelet dense granules results in a bleeding disorder of variable severity termed "storage pool defect" (SPD). We have examined the fawn-hooded hypertensive (FHH) rat as a model of SPD in order to genetically map the locus (Bd) responsible for prolonged bleeding. Platelet function assays of the FHH rat confirmed the presence of a platelet dense granule SPD. However electron microscopy and lysosomal enzyme assays indicated differences between the FHH rat and other rodent models of SPD. Genetic mapping through the use of congenic FHH rats localized the Bd locus to an approximately 1 cM region on rat chromosome 1. Through the use of comparative mapping between species and analysis of the initial draft of the rat genome assembly, six known and thirty-four putative genes were identified in the Bd locus. None of these genes have been previously implicated in platelet function. Therefore positional cloning of the gene responsible for the bleeding disorder in the FHH rat will lead to new insights in platelet physiology, with implications for diagnosis and management of hemostatic and thrombotic disorders.

Electron dense chains and clusters in human platelets.

Human platelets contain dense organelles that serve as storage sites for adenine nucleotides and serotonin. They also contain calcium and phosphate responsible for the opacity of dense bodies in the electron microscope. The present study has shown that there are also bead-like structures and clusters of beads that are electron opaque when viewed by the whole mount technique. Their origin and function are not known. However, their presence might interfere with the accuracy of imaging techniques currently being used to measure the number and volume of dense bodies in patients with platelet storage pool deficiency.

Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction.

BACKGROUND AND OBJECTIVES: Storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation. DESIGN AND METHODS: We included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting. RESULTS: Bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD. INTERPRETATION AND CONCLUSIONS: Tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins.

Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the gunmetal mouse.

The mutant gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky-Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue-specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue-specific effects of the gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.

A new genetic isolate of gray platelet syndrome (GPS): clinical, cellular, and hematologic characteristics.

Gray platelet syndrome (GPS) is a disorder characterized by thrombocytopenia and large platelets that lack alpha granules and their contents. We describe two siblings with GPS who are members of a Moslem Bedouin genetic isolate. The children, an 8-year-old girl and a 5-year-old boy, had characteristic pale blue platelets lacking alpha granules on electron microscopy. Platelet aggregation studies were normal. The girl underwent a bone marrow aspiration and biopsy which showed mild myelofibrosis and extensive emperipolesis, i.e., the passage of other hematopoietic cells through megakaryocytes. Both children lacked high-molecular-weight multimers of von Willebrand factor (vWF) and had reduced activity and antigens of vWf. Platelet activation was approximately normal when ADP was employed as agonist, but significantly impaired using the thrombin receptor-activating peptide (TRAP). These findings are explained in light of the mechanism of action of each agonist. In addition, we propose that the emperipolesis was caused by increased P-selectin in megakaryocytes, and resulted in release of fibroblastic growth factors, explaining the myelofibrosis. The detailed description of these cases provides a basis for future differentiation of the various types of GPS, and for our current attempts to isolate the gene causing GPS in this genetic isolate.