Diagnostic laboratory standardization and validation of platelet transmission electron microscopy.

Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

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.

The Medich giant platelet syndrome: two new cases.

Hypogranular platelet disorders in human subjects are relatively rare. They include the gray platelet syndrome, αδ storage pool deficiency, the Hermansky-Pudlak syndrome, and the white platelet syndrome. Perhaps the rarest of them all is the Medich giant platelet disorder. No additional cases of this condition have been reported since description of the first case in 2004. This study describes two children with thrombocytopenia and giant, hypogranular platelets found shortly after birth. Electron microscopic study of their platelets revealed sheets of membrane wrapped into tubes resembling scrolls. The scroll-like structures were open at both ends and often filled with glycogen particles. The abnormal structures are identical to those found in the initial case. As a result, the disorder can now be referred to as the Medich giant platelet syndrome.

The York platelet syndrome: a fourth case with unusual pathologic features.

The present report describes a fourth patient with platelet pathological features identical to those found in the first three cases with the York platelet syndrome (YPS), as well as other findings that suggest he may be a variant. His platelets contain the same giant opaque and target organelles found earlier, as well as enlarged organelles with a gray appearing matrix. It is possible that the giant structures have the same source, but are at different stages of development. The fourth patient has platelet pathology suggestive of other thrombocyte disorders. He has many large platelets and normal sized thrombocytes nearly devoid of alpha granules. As a result, he was originally thought to have the gray platelet syndrome. He also has significant numbers of platelets attached to platelets and platelets in platelets as seen in patients with the X-linked GATA-1 mutation. Some of the fourth YPS patient's platelets contained massive alpha granules suggesting the possibility of the Paris Trousseau Jacobson Syndrome. Yet, none of these other platelet disorders had giant dense organelles like those found in YPS thrombocytes. As a result, it is reasonable to include this child with the other three, and diagnose him as a patient with the YPS.

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.

Mice lacking the ITIM-containing receptor G6b-B exhibit macrothrombocytopenia and aberrant platelet function.

Platelets are highly reactive cell fragments that adhere to exposed extracellular matrix (ECM) and prevent excessive blood loss by forming clots. Paradoxically, megakaryocytes, which produce platelets in the bone marrow, remain relatively refractory to the ECM-rich environment of the bone marrow despite having the same repertoire of receptors as platelets. These include the ITAM (immunoreceptor tyrosine-based activation motif)-containing collagen receptor complex, which consists of glycoprotein VI (GPVI) and the Fc receptor γ-chain, and the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing receptor G6b-B. We showed that mice lacking G6b-B exhibited macrothrombocytopenia (reduced platelet numbers and the presence of enlarged platelets) and a susceptibility to bleeding as a result of aberrant platelet production and function. Platelet numbers were markedly reduced in G6b-B-deficient mice compared to those in wild-type mice because of increased platelet turnover. Furthermore, megakaryocytes in G6b-B-deficient mice showed enhanced metalloproteinase production, which led to increased shedding of cell-surface receptors, including GPVI and GPIbα. In addition, G6b-B-deficient megakaryocytes exhibited reduced integrin-mediated functions and defective formation of proplatelets, the long filamentous projections from which platelets bud off. Together, these findings establish G6b-B as a major inhibitory receptor regulating megakaryocyte activation, function, and platelet production.

Platelet-associated tissue factor enhances platelet reactivity and thrombin generation in experimental studies in vitro.

INTRODUCTION: The thrombogenic potential of tissue factor (TF) associated to platelets is controversial. We have investigated the in vitro contribution of platelet-associated TF to thrombus formation. MATERIALS AND METHODS: Platelets suspensions were exposed to human TF-rich microvesicles (TF-MV) from placental or recombinant origin. Platelet-associated TF was quantified through coagulometric assays. Adhesive and cohesive properties of platelets containing TF were assessed in perfusion models using two thrombogenic surfaces: 1) type-I collagen, or 2) damaged vascular segments. Perfusion studies were performed with heparinized blood enriched with a 30% of washed platelets exposed to TF-MV vs. washed control platelets. Thrombin generation and thromboelastometric properties of clots were also assessed using a fluorometric assay and ROTEM analysis, respectively. Inhibitory strategies with an antibody to TF were performed in some cases. RESULTS: The addition of 30% of platelets containing TF to blood perfusates resulted in a statistically significant increase in the platelet coverage (%CS) vs. non-exposed platelets on collagen surfaces (%CS: 19.7 ± 0.6 and 23.9 ± 0.7 respectively, vs.14.5 ± 1.4; p<0.01) and on the vascular subendothelium (%CS: 54.0 ± 1.5 and 47.2 ± 6.8 respectively vs. 38.0 ± 3.5, p<0.05), with a statistically significant increase in the size of large platelet aggregates (p<0.05) vs. control platelets. These effects on collagen surfaces were almost totally prevented by an antibody to TF. Platelet-associated TF significantly accelerated thrombin generation and clot formation (p<0.05), effects that were partially prevented by a neutralizing anti-TF. CONCLUSIONS: Platelet-associated TF potentiated adhesive and aggregating properties in in vitro studies with flowing blood and accelerated thrombin generation and clot formation time under steady conditions.

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.

Cytoskeletal F-actin, not the circumferential coil of microtubules, regulates platelet dense-body granule secretion.

Cytoskeletal F-actin assembly and microtubule reorganization are principal cellular events responsible for activation-induced platelet shape change; however, their roles in regulating platelet secretion have remained controversial. Herein, label-free microelectrochemistry techniques and pharmacological approaches are used to probe the role of F-actin and the microtubule in platelet dense-body secretion. Altered microtubule integrity via exposure to paclitaxel or vincristine had no effect on serotonin release in platelet suspensions. Disruption of F-actin by cytochalasin D (CytoD) or latrunculin A (LatA) substantially enhanced the rate of serotonin release, while inhibition of the F-actin-dependent platelet motor protein myosin IIA by blebbistatin had no effect. CytoD-treated platelets also showed enhanced serotonin quantal secretion rate. These results clearly indicate that F-actin, but not the microtubule, regulates platelet dense-body secretion and does so by serving as a physical barrier. This study also demonstrates the promise of microelectrochemistry for giving important insight into platelet quantal secretion mechanisms in future studies.

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.

NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is characterized by large platelets that lack α-granules. Here we show that mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain protein, cause GPS and that megakaryocytes and platelets from individuals with GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.

A BLOC-1 mutation screen reveals that PLDN is mutated in Hermansky-Pudlak Syndrome type 9.

Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patient's cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patient's melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boy's severe albinism and establishing his diagnosis as HPS-9.

Electrochemical measurement of endogenous serotonin release from human blood platelets.

Platelet aggregation in the bloodstream is tightly associated with the secretory function of platelets based on several types of cytoplasmic secretory granules, each sequestering distinct chemical messenger species. Dense-body granules are one prominent type of secretory granule responsible for storing small molecule chemical messengers. Upon platelet activation, the timely and rapid release of these small molecules is critical in facilitating platelet aggregation. Therefore, techniques capable of measuring real-time granule content release are needed to understand the fundamental properties of platelet secretion and aggregation. Existing techniques lack adequate time resolution or require potentially toxic exogenous reagents for real-time measurement of granule content release. Herein, we demonstrate a label-free electrochemical method based on the endogenous electroactive chemical messenger serotonin (5-hydroxytryptamine or 5-HT) for the real-time measurement of dense-body granule secretion from platelet suspensions; fast-scan cyclic voltammetry (FSCV) using carbon-fiber microelectrodes was chosen on the basis of its excellent temporal resolution, high sensitivity, and the ability to provide the electrochemical signature cyclic voltammograms for molecular identification. Real-time serotonin release from thrombin-stimulated human platelet suspensions was successfully measured, and the amount and time course of the bulk serotonin release were found to agree well with data obtained from single platelet measurements, thus confirming accurate characterization of granular secretion. Furthermore, this electrochemical method was applied to study the stimulation-secretion coupling in platelets, serotonin storage and release dynamics with applied pharmacological agents, and chemical messenger storage deficiency in Hermansky-Pudlak Syndrome (HPS) platelets, and the potential of this method to reveal secretion behavior in both normal and diseased platelets has clearly been demonstrated.