A novel mutation in a Turkish patient with Hermansky-Pudlak syndrome type 5

The Hermansky-Pudlak syndrome (HPS) is a rare genetically heterogeneous autosomal recessive disorder, characterized by tyrosinase-positive oculocutaneous albinism, platelet dysfunction and lysosomal ceroid lipofuscin storage. This is caused by defects in lysosome-related organelles. In humans eight different types of the syndrome are known, of which a short overview is given. The clinical features and a novel mutation of a patient with HPS type 5 are described here.     

Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.     

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.     

Hermansky-Pudlak syndrome subtype 5 (HPS-5) novel mutation in a 65 year-old with oculocutaneous hypopigmentation and mild bleeding diathesis: The importance of recognizing a subtle phenotype

Hermansky-Pudlak syndrome (HPS) ? characterized by the distinct clinical phenotypes of both oculocutaneous albinism and mild bleeding diathesis–is caused by mutations in genes that have crucial roles in the assembly of cellular organelles (skin melanosomes, platelet delta [dense] granules, lung lamellar bodies, and cytotoxic T-cell lymphocyte granules). Immunodeficiency, pulmonary fibrosis and granulomatous colitis are associated with some, but not all subtypes of HPS, with varying degrees of clinical severity. We describe a patient diagnosed with platelet dense granule storage pool deficiency (DG-SPD) at age 38 years after he presented with spontaneous intracranial hemorrhage. His mild oculocutaneous hypopigmentation was subtle. In the following 27 years, he did not develop severe bleeding nor pulmonary or gastrointestinal complications. A novel homozygous c.1960A>T; p.Lys654* mutation in the HPS-5 protein gene (HPS5) was identified through next generation sequencing, (NGS) which is consistent with the patient’s clinical and laboratory phenotypes. This case underscores the importance of recognizing the mild clinical phenotype of HPS-5 and utilization of both laboratory and molecular testing for diagnosis, prognostication, and surveillance for end organ damage in patients affected with HPS.     

Studies on a new variant of the Hermansky-Pudlak syndrome: Qualitative,ultrastructural, and functional abnormalities of the platelet-dense bodies associated with a phospholipase a defect

The structure and functions of platelets from a patient in whom albinism and hemorrhagic diathesis were associated have been investigated. Electron microscope studies showed a large reduction in the number of dense bodies and this was confirmed by an examination of fluorescent platelets loaded with mepacrine. The rare dense bodies were much bigger than normally observed; their density was diminished and was localized in a peripheral ring. Other platelet constituents were found to be normal. Platelet peroxidase activity was normal in the canaliculi of the dense tubular system; catalase-positive granules were also present. Serotonin uptake by the patient's platelets was much decreased and reserpine, a potent inhibitor of serotonin accumulation by normal human platelets, did not further decrease this incorporation. The uptake of free ¹⁴C-arachidonic acid by the platelets was greatly diminished, as was its thrombin-induced liberation from phosphatidylcholine and phosphatidyl inositol. Moreover, platelet phospholipase A₁ activity was much reduced and phospholipase A₂ activity was undetectable.     

Increased platelet Fc receptor expression as a potential contributing cause of platelet hypersensitivity to collagen in diabetes mellitus

Recent clinical and laboratory observations support a potential role for the platelet FcgammaRIIA receptor (FcR) in collagen-mediated platelet activation associated with arterial thrombosis. Stable age- and sex-independent variation in receptor expression exists. Flow cytometry showed that 100 diabetes patients had increased mean platelet FcR expression levels compared with 201 non-diabetes patients (P < 0.001). Aggregation studies following FcR cross-linking supported a receptor role in increased activation of diabetes platelets after collagen stimulation (P = 0.018). Immunoprecipitation of collagen-stimulated and FcR-crosslinked platelets demonstrated enhanced levels of tyrosine-phosphorylated Syk in diabetic platelets. Increased platelet FcR-mediated sensitivity to collagen may contribute to cardiovascular morbidity and mortality in diabetes.     

Daily exposure to forty percent oxygen causes a decrease in platelet count

A study was undertaken to evaluate a previous observation made in this laboratory that hyperoxic treadmill training is associated with a decrease in the circulating platelet count (PLT). The subjects studied breathed air containing 40% oxygen for 30 min/day, 5 days/week, for 6 weeks, while at rest in the seated position. Such exposure resulted in a consistent fall in PLT between day 1 and day 5 of each week (P less than .01). In addition, there was a progressive decline in PLT over the 6 weeks of exposure to the hyperoxic air, PLT being inversely correlated with the duration of hyperoxic exposure (rs = -0.886, P less than .02). The total decrease in PLT over the 6 weeks was 56 +/- 46 x 10(9) l(-1) (P less than .025). Not surprisingly, these changes were mirrored in the plateletcrit (Pct). The decrease in PLT did not appear to be secondary to either hemodilution or reduced erythropoietic stimulation. The mechanism of production and the biological significance of these changes remain to be elucidated.     

Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantation

Reticulated platelets (RP) are the youngest forms of platelets in blood and reflect the rate of bone marrow platelet production. In the present study, we used flow cytometric analysis to determine the percentage of RPs in patients undergoing allogeneic stem cell transplantation. We investigated 10 patients after transplantation from HLA identical siblings: five with acute myeloid leukemia (AML), four with chronic myeloid leukemia (CML), and one patient with myelodysplastic syndrome (MDS). Of the patients examined, four patients underwent allogeneic bone marrow transplantation and six patients underwent peripheral blood stem cell transplantation. It was observed that the initially reduced percentage of RPs (2.9 +/- 1.7%; mean +/- SD) was significantly higher (P = 0.0109) in all patients (13.6 +/- 6.4%) in the following 10-26 days. The RP percentage peak preceded the recovery of peripheral platelet count up to 45.6 x 10(9)/l on average by 3 days. We found no difference in RP% between the AML and CML patients but we did observe that in CML patients the RP percentage increased on average 7 days earlier than in AML patients. The elevated RP percentage reflects increased bone marrow regeneration and can be considered an additional marker of thrombopoietic recovery in the patients undergoing allogeneic stem cell transplantation.     

IgG from patients with antiphospholipid syndrome binds to platelets without induction of platelet activation

The clinical manifestations of the antiphospholipid syndrome (APLS) include arterial and venous thrombosis, thrombocytopenia and fetal loss, but the pathogenic mechanisms remain unclear. It has been hypothesized that platelet activation by autoantibody may be a pathogenic mechanism. We studied IgG binding, microparticle (mp) formation and P-selectin expression by flow cytometry in normal platelets after incubation in serum from 11 patients with antiphospholipid antibodies and that from 10 normal healthy subjects. Levels of platelet-associated IgG were significantly higher after incubation in patient sera (mean 17.2, range 2.0–75.0%) compared with normal sera (mean 2.0, range 1.2–3.7%, P  < 0.05). Incubation of normal platelets in serum led to increased microparticle formation ( P  < 0.01) and P-selectin expression ( P  < 0.05), compared with unstimulated platelets. There was no significant difference, however, between microparticle formation nor P-Selectin expression induced by patient serum (mp 3.0 (1.6–5.0)%; P-selectin 8.0 (4.0–16.6)%) versus normal serum (mp 3.2 (2.1–4.5)%; P-selectin 10.1 (4.0–15.6); median (range)). Pre-activation of platelets with subthreshold ADP concentrations or thrombin receptor activator peptide resulted in a small increase in microparticle formation, but there was still no significant difference between the effects of patient and control sera. Despite the presence of platelet membrane binding IgG in serum from 5/11 patients with antiphospholipid antibodies, there was no evidence for associated enhanced platelet-activating ability. This study supports antiplatelet reactivity in antiphospholipid syndrome, but not a direct platelet-activating role for platelet-directed autoantibodies.     

Extracts from Trifolium pallidum and Trifolium scabrum aerial parts as modulators of blood platelet adhesion and aggregation

A growing number of reports indicate that some species of clover (Trifolium) may have remarkable medical importance; however, the effects of these plants on blood platelets and hemostasis are inadequately recognized. This work was designed to study the effects of Trifolium pallidum and Trifolium scabrum extracts on the functions of human blood platelets in vitro. Platelet suspensions were preincubated with extracts from aerial parts of T. pallidum (phenolic fraction and clovamide fraction) and T. scabrum (phenolic fraction) at the final concentrations of 12.5, 25, and 50?µg/ml. Then, for platelet activation thrombin (0.1?U/ml), thrombin receptor activating peptide (TRAP; 20?µM), or adenosine diphosphate (ADP; 1?µM) were used. The effects of Trifolium extracts on adhesion of blood platelets to fibrinogen and collagen were determined by enzyme-linked immunosorbent assay (ELISA) method. Platelet aggregation was monitored on a dual-channel Chronolog aggregometer. In these studies, we also compared the action of tested plant extracts with the effects of another antiplatelet plant-derived compound – resveratrol (3,4′,5-trihydroxystilbene). The performed assays demonstrated that the tested extracts might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of all tested extracts on adhesion of thrombin-stimulated platelets to collagen was found. Both extracts from T. pallidum and from T. scabrum reduced the thrombin-induced platelet adhesion to fibrinogen. Furthermore, in the presence of all three extracts, the platelet aggregation induced by thrombin was slightly inhibited. Our results also indicate that the tested plant extracts (at the highest concentrations used of 50?µg/ml), similar to purified resveratrol, inhibit selected steps of platelet activation stimulated by both proteolytic (thrombin) and nonproteolytic agonists (TRAP or ADP). In the comparative studies, T. pallidum and T. scabrum extracts was not found to be more effective antiaggregatory factor, than resveratrol. Extracts from T. pallidum and T. scabrum aerial parts reveal antiplatelet properties: the antiadhesive effect was similar to that of the reference compound resveratrol, whereas the antiaggregant effect was less marked. The results obtained suggest that these plants may be a promising source of natural compounds, valuable in the prevention of the enhanced activity of blood platelets in numerous cardiovascular diseases, observed in menopausal or postmenopausal women.     

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

Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.     

Acetaminophen and diphenhydramine as premedication for platelet transfusions: a prospective randomized double-blind placebo-controlled trial

Non-hemolytic transfusion reactions (NHTR) occur in up to 30% of patients receiving platelet transfusions. Premedication with acetaminophen and diphenhydramine is a common strategy to prevent NHTR, but its efficacy has not been studied. In this prospective trial, transfusions in patients receiving pre-storage leukocyte-reduced single-donor apheresis platelets (SDP) were randomized to premedication with either acetaminophen 650 mg PO and diphenhydramine 25 mg IV, or placebo. Fifty-one patients received 98 transfusions. Thirteen patients had 15 NHTR: 15.4% (8/52) in the treatment arm and 15.2% (7/46) in the placebo arm. Premedication prior to transfusion of pre-storage leukocyte reduced SDP does not significantly lower the incidence of NHTR as compared to placebo.     

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

Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.     

Usefulness of the epicardial fat tissue thickness as a diagnostic criterion for geriatric patients with metabolic syndrome

Objective To evaluate the epicardial fat tissue thickness (EFTT) as a diagnostic criterion for geriatric patients with metabolic syndrome (MetS). Methods Sixty geriatric patients over 65 years of age were recruited for the study. Patients were divided into two groups: Group 1 (n = 30) consisted of patients with MetS; Group 2 (n = 30) consisted of patients without MetS. Echocardiography was used to measure EFTT in all patients, and blood samples were analyzed for biochemical parameters. Results Compared to Group 2, EFTT levels of Group 1 were statistically higher (P < 0.05). In a binary logistic regression analysis, EFTT levels served as the independent factor for metabolic syndrome (B = 17.35, SE = 4.93, Wald = 12.36, P < 0.001). Receivers operating characteristic Curve (ROC-curve) analysis revealed that EFTT predicted MetS with 96.7% sensitivity and 86.7% specificity above the level of 7.3 mm [area under the curve = 0.969; 95% confidence interval (CI): 0.928–1.00]. Conclusions The present study demonstrated that serum EFTT levels were higher in geriatric patients with MetS and can therefore be used as a diagnostic criterion for MetS.     

Sleep apnea syndrome. A critical review of the apnea index as a diagnostic criterion

The utility of the apnea index (number of apneic events per hour of sleep) in diagnosing sleep apnea syndrome is reviewed. Data from currently extant reports indicate that many otherwise normal, aging subjects may be classified as having sleep apnea syndrome based on the current diagnostic threshold of five apneic episodes per hour: A chi 2 analysis suggests a relationship between age and level of sleep apnea. Several other reports indicate that use of a threshold of five apneic episodes per hour does not reliably predict increased health risk or somnolence in aging subjects. Adjustment of the apnea index, based on studies of aging normal subjects and of aging patients with sleep apnea syndrome is necessary to ensure reliable results in clinical and research applications.     

Resveratrol,a phenolic antioxidant with effects on blood platelet functions

The main purpose of this article is to provide an overview of the currently available evidence of antiplatelet properties of resveratrol (3,4^′,5-trihydroxystilbene). Resveratrol, a phenolic compound found naturally in fruits, nuts, flowers, seeds and bark of different plants is integral part of human diet. It exhibits a wide range of biological effects, including antiplatelet, anti-inflammatory, anticancer, antimutagenic and antifungal properties. It is also a potent antioxidant, reactive oxygen species scavenger and metal chelators. Resveratrol reduces lipid peroxidation, oxidation and nitration of platelet and plasma proteins. This review article describes the chemical structure of resveratrol, its biological activity, the effects on blood platelet functions and the mechanisms involved in its action on blood platelets, the cells which play an important role not only in the haemostatic process, but also in pathogenesis of cardiovascular diseases.     

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.     

Deficiency of P-selectin in a patient with grey platelet syndrome

Abstract: Patient B.G. is a 29-yr-old female with a lifelong bleeding disorder characterized clinically by a highly increased bleeding time, menorrhagias, long-lasting bleeding after cuts and tooth extractions and large post-traumatic haematomas. Her coagulation tests were within normal range, platelet count was 140,000–160,000 per μl, but platelet function was impaired as demonstrated by the absence of collagen-induced aggregation, although no abnormalities were detected in aggregation response to ADP and ristocetin. Morphologically her platelets were characterized by gigantic size – average profile area was about 2.5 times higher than that of control donors, and severe deficiency of α-granules – only 16% of their number in control donors. These features taken together indicated the diagnosis of grey platelet syndrome. As has been shown by quantitative immunoblotting, patient's platelets contained small amounts of α-granule membrane protein P-selectin – about 15% of that in control donors. The content of plasma membrane glycoproteins IIb–IIIa and Ib was not reduced, suggesting the specific deficiency of α-granule membrane protein. Thus, B.G. is the second patient described in the literature (see also Lages et al, J Clin Invest 1991: 87: 919–929) with combined deficiency of α-granules and P-selectin.     

The impact of autologous platelet concentrates on endodontic healing: a systematic review

The current literature was reviewed to determine the impact of autologous platelet concentrates (APCs) on endodontic healing. All types of clinical study designs concerning any kind of endodontic treatment involving the application of APCs were included. Two independent reviewers searched three databases (PubMed, Embase, and Cochrane Library) for studies, complemented by hand search, until 16/1/2016. From the 423 identified records, 48 articles met the inclusion criteria. Selected randomized controlled clinical trials (RCTs) underwent Cochrane Collaboration’s risk-of-bias assessment and data extraction. Only two RCTs showed low risk of bias. There was considerable heterogeneity between the RCTs with regard to the type of therapy, type of APCs, assessment method, and study quality, and therefore the data could not be analyzed quantitatively. The included case reports/series and non-randomized comparative studies underwent qualitative analysis with the revised Methodological Index for Non-Randomized Studies (MINORS) and data extraction. The two comparative non-randomized studies scored qualitatively high, though the MINORS-scores of the case series and reports were dispersed. APCs were involved in five endodontic treatment modalities, namely apexification, regenerative endodontic procedures, pulpotomy, apical surgery, and treatment of endo-perio/perio-endo lesions. APCs seem to accelerate postoperative bone healing, augment the patients’ postoperative quality of life, aid further root development, and support maintenance/regaining of pulp vitality. No adverse events were reported. APCs in endodontic treatments seem to contribute to the healing of soft and hard tissues, though there is a lack of long-term high quality clinical trials and standardized treatment protocols.