York platelet syndrome is a CRAC channelopathy due to gain-of-function mutations in STIM1.

Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS.

Hermansky-Pudlak syndrome (HPS5) in a nonagenarian.

Hermansky-Pudlak syndrome (HPS) is an autosomal-recessive disorder clinically characterized by oculocutaneous albinism, bleeding diatheses, and lysosomal accumulation of ceroid lipofuscin, which in some cases may cause granulomatous colitis and pulmonary fibrosis. Any of these complications could result in a shortened life span for patients with HPS. We report a 92-year-old man with HPS 5 who, to our knowledge, is the oldest patient with HPS documented in the literature. This report highlights the importance of typing HPS to counsel patients regarding disease prognosis.

Rapid ultrastructural detection of success or failure after bone marrow transplantation in the Chediak-Higashi syndrome.

The present study has used electron microscopic techniques to rapidly detect the success or failure of bone marrow transplantation in three patients with the Chediak-Higashi Syndrome (CHS). The most rapid procedure was the whole mount technique to determine the presence or absence of dense bodies, which are inherently electron-opaque, serotonin-containing storage organelles in platelets. Dense bodies were present in normal numbers in platelets from two patients with successful transplantation and absent in thrombocytes from another patient in whom the transplant had failed.

Platelet IκB kinase-ß deficiency increases mouse arterial neointima formation via delayed glycoprotein Ibα shedding.

OBJECTIVE: On the luminal surface of injured arteries, platelet activation and leukocyte-platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor-κB is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-κB pathway in forming arterial neointima after arterial injury. METHODS AND RESULTS: We performed carotid artery wire injuries in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice with a platelet-specific deletion of IκB kinase-ß (IKKß) (IKKß(fl/fl)/PF4(cre)/LDLR(-/-)) and in control mice (IKKß(fl/fl)/LDLR(-/-)). The size of the arterial neointima was 61% larger in the IKKß(fl/fl)/PF4(cre)/LDLR(-/-) mice compared with the littermate control IKKß(fl/fl)/LDLR(-/-) mice. Compared with the control mice, the IKKß(fl/fl)/PF4(cre)/LDLR(-/-) mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ibα shedding after platelet activation was compromised in the IKKß-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17, the key enzyme involved in mediating glycoprotein Ibα shedding in activated IKKß-deficient platelets. CONCLUSIONS: Platelet IKKß deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-κB-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.

Blood-nanoparticle interactions and in vivo biodistribution: impact of surface PEG and ligand properties.

Theranostic nanoparticles (NPs) cannot reach their target tissue without first passing through blood; however, the influence of blood protein and blood cell interactions on NP biodistribution are not well understood. The current work shows that 30 nm PEGylated gold NPs (GNPs) interact not only with blood proteins as thought before but also with blood cells (especially platelets and monocytes) in vivo and that longer blood circulation correlates strongly with tumor uptake. Further, GNP surface properties such as negative charge or lyophilization had either a minimal (i.e., charge) or 15-fold increase (i.e., fresh vs lyophilized) in blood retention times and tumor uptake. Tumor accumulation was increased over 10-fold by use of a bioactive ligand (i.e., TNF) on the lyophilized GNP surface. Resident macrophages were primarily responsible for the bulk of GNP uptake in liver while spleen uptake was highly surface property dependent and appears to involve macrophages and cellular interaction between the red and white pulp. This study shows that the PEG layer and ligand on the surface of the NP are critical to blood interactions and eventual tumor and RES organ biodistribution in vivo.

A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.

Clinical, molecular, and cellular features of non-Puerto Rican Hermansky-Pudlak syndrome patients of Hispanic descent.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive condition characterized by a bleeding diathesis and hypopigmentation of the skin, hair, and eyes. Some HPS patients develop other complications such as granulomatous colitis and/or fatal pulmonary fibrosis. Eight genes have been associated with this condition, resulting in subtypes HPS-1 through HPS-8. The HPS gene products are involved in the biogenesis of specialized lysosome-related organelles such as melanosomes and platelet delta granules. HPS1 and HPS4 form a stable complex named biogenesis of lysosome-related organelles complex (BLOC)-3, and patients with BLOC-3 or AP-3 deficiency develop pulmonary fibrosis. Therefore, it is important to subtype each HPS patient. HPS type 1 (HPS-1) occurs frequently on the island of Puerto Rico because of a founder mutation. Here, we describe seven mutations, six of which, to our knowledge, are previously unreported in the HPS1, HPS4, and HPS5 genes among patients of Mexican, Uruguayan, Honduran, Cuban, Venezuelan, and Salvadoran ancestries. Our findings demonstrate that the diagnosis of HPS should be considered in Hispanic patients with oculocutaneous albinism and bleeding symptoms. Moreover, such patients should not be assumed to have the HPS-1 subtype typical of northwest Puerto Rican patients. We recommend molecular HPS subtyping in such cases, as it may have significant implications for prognosis and intervention.

Homozygosity mapping with SNP arrays confirms 3p21 as a recessive locus for gray platelet syndrome and narrows the interval significantly.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Chediak-Higashi syndrome with early developmental delay resulting from paternal heterodisomy of chromosome 1.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable oculocutaneous albinism, immunodeficiency, mild bleeding diathesis, and an accelerated lymphoproliferative state. Abnormal lysosome-related organelle membrane function leads to the accumulation of large intracellular vesicles in several cell types, including granulocytes, melanocytes, and platelets. This report describes a severe case of CHS resulting from paternal heterodisomy of chromosome 1, causing homozygosity for the most distal nonsense mutation (p.E3668X, exon 50) reported to date in the LYST/CHS1 gene. The mutation is located in the WD40 region of the CHS1 protein. The patient's fibroblasts expressed no detectable CHS1. Besides manifesting the classical CHS findings, the patient exhibited hypotonia and global developmental delays, raising concerns about other effects of heterodisomy. An interstitial 747 kb duplication on 6q14.2-6q14.3 was identified in the propositus and paternal samples by comparative genomic hybridization. SNP genotyping revealed no additional whole chromosome or segmental isodisomic regions or other dosage variations near the crossover breakpoints on chromosome 1. Unmasking of a separate autosomal recessive cause of developmental delay, or an additive effect of the paternal heterodisomy, could underlie the severity of the phenotype in this patient.

Hermansky-Pudlak syndrome in two African-American brothers.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and, in some patients, granulomatous colitis and/or a fatal pulmonary fibrosis. There are eight different subtypes of HPS, each due to mutations in one of eight different genes, whose functions are thought to involve intracellular vesicle formation and trafficking. HPS has been identified in patients of nearly all ethnic groups, though it has primarily been associated with patients of Puerto Rican, Northern European, Japanese and Israeli descent. We report on the diagnosis of HPS type 1 in two African-American patients. Both brothers carried compound heterozygous mutations in HPS1: previously reported p.M325WfsX6 (c.972delC) and a novel silent mutation p.E169E (c.507G > A), which resulted in a splice defect. HPS may be under-diagnosed in African-American patients and other ethnic groups. A history of easy bruising or evidence of a bleeding disorder, combined with some degree of hypopigmentation, should prompt investigation into the diagnosis of HPS.

External calcium facilitates signalling, contractile and secretory mechanisms induced after activation of platelets by collagen.

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca2+. We have studied the involvement of exogenous Ca2+ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca2+ levels ([Ca2+]i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca2+ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca2+, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca2+]i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca2+. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca2+, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca2+ depends on a functional cytoskeleton.

Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.

Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG-/- mice. Wild-type (WT) but not SG-/- platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal alpha-granule structure in SG-/- platelets. However, SG-/- platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG-/- megakaryocytes showed extensive emperipolesis of neutrophils. SG-/- platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG-/- platelets. The alpha-granule proteins platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG-/- platelets. Exposure of P-selectin and alphaIIb after thrombin treatment was similar in WT and SG-/- platelets. SG-/- mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG-/- platelet function deficiencies are related to inadequate packaging and secretion of selected alpha-granule proteins and reduced secretion of dense granule contents critical for platelet activation.

Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa.

We investigated the interactions of vesicles containing human tissue factor (TF) with platelets and evaluated responses induced by rFVIIa using standard aggregometry, ultrastructural and flow-cytometry techniques. Washed platelets were exposed to a preparation of placental human TF (pTF) or to a relipidated formulation of recombinant human TF (rTF). Under stirring conditions, pTF induced reversible aggregation with platelets returning to their resting state after 5 minutes. This reversible response to pTF was partially inhibited by antibodies against CD62-P, but not by antithrombin agents, and was not observed with rTF. Sequential ultrastructural studies revealed uptake of both TF preparations by platelets involving traffic of vesicles through channels of the open canalicular system (OCS). Immunocytochemical studies on cryosections identified TF in the OCS, and occasionally in the alpha-granules of the platelets. These processes were faster with pTF than with rTF, but both TF preparations accumulated in platelets at the end of incubation periods. Flow cytometry studies revealed the presence of other cellular antigens (CD62-P, CD14 and CD45) associated to the pTF. Addition of rFVIIa to washed platelets exposed to pTF or rTF, caused a thrombin dependent irreversible platelet aggregation. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes are accelerated by the presence of other cellular antigens in the vesicles. Our findings may explain the hemostatic action of rFVIIa in severely hemodiluted patients, but are also relevant for the understanding of potential implications of TF-associated to platelets in the propagation of thrombus.

Intestinal disease in Hermansky-Pudlak syndrome: occurrence of colitis and relation to genotype.

BACKGROUND & AIMS: Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder characterized by oculocutaneous albinism and platelet dysfunction, results from mutations in 1 of at least 7 different genes. Some patients develop a fatal pulmonary fibrosis and others a disabling colitis. This study aimed to document the occurrence of colitis among HPS patients, characterize gastrointestinal tract involvement in HPS, and analyze the distribution of colitis among HPS genotypes. METHODS: Of the 122 HPS patients followed at the National Institutes of Health Clinical Center between 1993 and 2005, 24 were evaluated by endoscopy for gastrointestinal complaints. The histology of gastrointestinal biopsies was retrospectively examined to assess for inflammatory changes, granulomata, and pigmented macrophages. These data were compared with symptoms and HPS genetic subtypes. RESULTS: At colonoscopy, 7 of 23 patients (30%) had endoscopic mucosal abnormalities, including nodularity, erythema, petechiae, or erosions. Six of these 7 patients (86%) had findings of colitis on biopsy. Of the 16 patients with normal-appearing colonic mucosa, 2 patients (12%) had colitis on biopsy. Pigmented macrophages were also observed in the colonic lamina propria in 16 of the 23 patients (70%). Of the 8 patients with confirmed colitis, 7 had the HPS-1 subtype, and 1 had the HPS-4 subtype. CONCLUSIONS: There is an increased frequency of colitis in our population of 122 HPS patients (8/122, 7%) and in HPS patients referred specifically for symptom evaluation (8/24, 33%). Colitis was found in patients with HPS-1 and HPS-4 genotypes.

Successful bilateral lung transplantation for pulmonary fibrosis associated with the Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by oculocutaneous albinism, a bleeding diathesis, and in a subset of patients, pulmonary fibrosis. Lung transplantation, the only curative therapy for pulmonary fibrosis, has not been previously reported as a successful treatment strategy for patients with HPS because the bleeding diathesis was thought to contraindicate major thoracic surgery. We successfully performed bilateral sequential lung transplantation in a patient with pulmonary fibrosis and HPS after transfusion of 6 units of platelets. Lung transplantation is a viable therapeutic option in patients with pulmonary fibrosis and only a mild bleeding diathesis associated with HPS.

Cellular, molecular and clinical characterization of patients with Hermansky-Pudlak syndrome type 5.

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelles such as melanosomes and platelet dense granules. Seven genes are now associated with HPS in humans. An accurate diagnosis of each HPS subtype has important prognostic and treatment implications. Here we describe the cellular, molecular, and clinical aspects of the recently identified HPS-5 subtype. We first analyzed the genomic organization and the RNA expression pattern of HPS5, located on chromosome 11p14, and demonstrated tissue-specific expression of at least three alternatively spliced HPS5 mRNA transcripts, coding for HPS5A and HPS5B proteins, that differ at their 5'-ends. Genetic screening of 15 unassigned HPS patients yielded six new HPS5 mutations in four patients. Clinically, our HPS-5 patients exhibited iris transillumination, variable hair and skin pigmentation, and absent platelet dense bodies, but not pulmonary fibrosis or granulomatous colitis. In two patients with homozygous missense mutations, hemizygosity was ruled out by gene-dosage multiplex polymerase chain reaction, and immunocytochemical analyses of their fibroblasts supported the HPS-5 diagnosis. Specifically, LAMP-3 distribution was restricted to the perinuclear region in HPS-5 fibroblasts, in contrast to the normal LAMP-3 distribution, which extended to the periphery. This specific intracellular vesicle distribution in fibroblasts, in combination with the clinical features, will improve the characterization of the HPS-5 subtype.

Giant electron dense chains, clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder III. Platelet analytical electron microscopy.

A woman and her son were referred because of prolonged thrombocytopenia. Ultrastructural studies revealed the presence of giant organelles in their cells never observed previously in human platelets. Our initial reports described the evolution of two types of giant organelles in patient megakaryocytes and platelets. The last report also demonstrated that the large organelles in platelet whole mount preparations were inherently electron opaque like serotonin-rich dense bodies in normal and patient platelets. In the present study analytical electron microscopy of whole mount preparations from the patients and controls revealed that the inherently electron opaque dense and hexagonal precursor fragments, chains, clusters and giant organelles contained high concentrations of calcium and phosphorous, the major elements in normal dense bodies. The ratio of calcium to phosphorous in patient giant organelles was nearly the same as in normal platelet dense bodies. However, as shown in the previous study, levels of serotonin and adenine nucleotides were normal in patient platelets, and giant organelles and their contents were not secreted following activation of platelets with thrombin. Thus, despite the similarity in mineral content responsible for electron density, the giant organelles in patient platelets do no appear to be aberrant serotonin storage organelles.

Giant electron-dense chains, clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder.

A woman and her male child were referred because of life-long thrombocytopenia, moderately increased platelet size, and absence of laboratory findings suggesting immune thrombocytopenia or defective platelet function. Evaluation of their platelets in the electron microscope revealed the presence of large organelles never seen before in human platelets. Examination of bone marrow from the mother and her son in an earlier study revealed that the giant platelet organelles originated in megakaryocytes. The present study has focused on the continuing development of the aberrant organelles in circulating platelets. The smallest subunits were electron-dense fragments and hollow-cored bodies observed in the dense tubular system (DTS). The dense fragments formed chains that became thicker, resulting in clusters, and clusters formed the large electron opaque bodies. Hollow-cored, almost hexagonal subunits also formed chains that interacted with each other to form target-like organelles. The multi-layered target organelles tended to become completely electron dense and difficult to distinguish from the opaque bodies. How two different types of aberrant organelles can develop in the same megakaryocyte/platelet system and both originate from channels of the DTS is unknown. Partial clarification stemming from analytical electron microscopy and ultrastructural cytochemistry will be presented in a subsequent report.