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Introduction The pathogenesis of heparin-induced thrombocytopenia (HIT) primarily involves anti-platelet factor(PF)4/heparin antibodies engaging FcγRIIa receptors on platelets to trigger a deleterious cascade of activation, thrombosis, and thrombocytopenia. Recent work with murine monoclonal antibodies has shed light on epitopic determinants that potentiate platelet activation (pathogenic KKO vs non-pathogenic RTO antibodies), but these mechanisms may not fully reflect the gamut of polyspecific antibodies seen in human clinical practice. We examined the development and characteristics of anti-PF4/heparin antibodies in patients undergoing cardiac surgery. Methods After obtaining informed consent from patients undergoing cardiac bypass surgery, we performed ELISA using: (1) commercial Diagnostica Stago Asserachrom IgG kit (Cat.Nr 00624); or (2) in-house ELISA with platelet derived native(n)PF4. ELISA performed as per manufacturer instructions and published methods. Bacterial genomic nucleic acids (NA) were extracted from overnight culture of BL21(DE3)pLysS-wtPF4 using Promega kit Wizard® SV Genomic DNA Purification System (#A2361, Promega). Cross linking assays were performed as previously described using Bis(sulfosuccinimidyl)suberate (BS3) obtained commercially (#21585, Thermo Scientific Pierce). Optical density was normalised with controls to assist interassay comparability. All samples were screened with the commercial kit day 7 post-operatively, or the nearest available serum between day 4-10. Positive samples were then examined longitudinally to chart the onset of antibody formation and their duration. Results Commercial ELISA identified 30/123 (24%) patients positive for anti-PF4/heparin antibodies following cardiac surgery, but there was no HIT observed in the complete cohort of 123 patients and therefore all 30 were false positive. Surprisingly, in-house ELISA using nPF4/heparin was negative for 22/30, but the addition of NA to PF4 rescued the OD of these to become positive again. In contrast, OD fell for all true HIT controls with the addition of NA, as they did for a subset of the false-positives 7/30 [Figure A]. Five patients had persistently raised OD despite the addition of NA, suggesting the presence of both NA/nPF4/heparin antibodies and nPF4/heparin antibodies. Overall, the incidence of false-positive anti-PF4/heparin antibodies was only 12/123 (10%) using the in-house ELISA method with nPF4. SRA was performed on all patients with nPF4/heparin antibodies, but they were all negative. Spiking nPF4 with increasing concentrations of NA caused reduction of tetramer formation and an increasing proportion of dimeric and monomeric forms seen on cross-linkage assays [Figures B and C]. Longitudinal studies demonstrated that all 13 patients positive by commercial ELISA with only low range absolute results (OD Conclusions Increasing amounts of NA inhibit PF4 tetramer formation, and reduce the ability for HIT antibodies to bind. Thus, NA may participate in the preferential presentation of PF4 monomers that are recognised by non-pathogenic NA/PF4 monomer/heparin antibodies, similar to RTO. There is a smaller subset of "true" non-pathogenic antibodies that bind to PF4 tetramers, and an even smaller number of patients who may possess both classes of non-pathogenic antibodies. We speculate that pre-formed antibodies to NA/PF4 monomers are responsible for the majority of false positive results from commercial ELISA kits, and these are unrelated to the pathogenic anti-PF4/heparin antibodies of HIT. Figure Caption A: Representative example of five patients with increasing ELISA OD in the presence of NA (NA1-5), two patients with OD that fell in the presence of NA (TFP1-2), and known HIT serum with reduced OD in the presence of NA. B: Reduction in PF4 tetramer formation and increasing PF4 monomers with increasing NA concentration. Lanes from left-right: control PF4 without BS3, and then increasing concentration of NA from 0, 5, 30 and 45ng/μL with BS3. C: Percentage of PF4 as tetramers or monomers with increasing concentration of NA. Disclosures No relevant conflicts of interest to declare.

Adverse drug effects are common in clinical practice and often have a negative impact on patient safety. Heparin and heparin-derived drugs may induce an immune reaction, termed heparin-induced thrombocytopenia (HIT). HIT is mediated by IgG antibodies with specificity for heparin/platelet factor 4 (PF4) antigenic complexes. HIT is a hypercoagulable state which often causes severe and extensive thrombosis that results in high morbidity and mortality. The prevailing view is that these immune complexes activate platelets via FcγRIIa receptors leading to thrombocytopenia and thrombosis. Neutrophil extracellular traps (NETs) are DNA-containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non-immune disorders. Here we show that HIT immune complexes directly activate neutrophils via FcγRIIa and induce NETs formation. In addition, NETosis is also induced by activated platelets/neutrophil interactions mediated by P-selecting and PSGL-1. Ex vivo reconstitution of the HIT condition in a microfluidics system demonstrated the formation of thrombi rich in platelets, neutrophils, extracellular DNA and citrullinated histone 3 (Figure 1A, left panels). Neutrophil depletion abolished thrombus formation. Conversely neutrophil reconstitution restored thrombus deposition (Figure 1A, middle panels and right panels). Moreover, neutrophils alone treated with HIT IgG plus heparin formed thrombi containing extracellular DNA networks and citrullinated histone 3 on P-selectin coated channels (Figure 1B). Establishment of HIT in hFcγRIIa+/hPF4+ transgenic mice (HIT mice) using HIT patient's IgG or the HIT-like monoclonal antibody KKO recapitulated the hallmarks of NETosis: citrullinated histone 3, cell free DNA and MPO were detected in plasma and the presence of neutrophils, extracellular DNA and citrullinated histone 3 was found in lung thrombi. Low density neutrophils were also present in HIT mice treated with HIT IgG plus heparin but not in animals treated with control IgG. Treatment of HIT mice with DNase I or NETs formation inhibition with the PAD4 inhibitor GSK484 led to a dramatic decrease in thrombosis. This was corroborated by deletion of PAD4 in HIT mice. No thrombi were detected in hFcγRIIa+/hPF4+/PAD4-/- mice treated with HIT IgG and heparin (Figure 1C), indicating that NETs formation is required for thrombosis. Unlike thrombosis, thrombocytopenia was not affected by the absence of NETs formation, suggesting that these are separable processes. However, they are FcγRIIa-mediated mechanisms as anti-FcγRIIa antibodies abolished both processes. Analyses of sera from HIT patients revealed the presence of NETs markers and a significant proportion of neutrophils from patients with active HIT were undergoing NETosis. Our observations demonstrate that NETs formation is present in HIT and that it is essential for the development of thrombosis. Thrombocytopenia is not affected by the absence of NETosis. These findings suggest a new concept of the pathogenesis of thrombosis in HIT and as such are of clinical significance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Romiplostim, a thrombopoietin mimetic, increases platelet counts in patients with immune thrombocytopenia, with few adverse effects. Methods In this open-label, 52-week study, we randomly assigned 234 adult patients with immune thrombocytopenia, who had not undergone splenectomy, to receive the standard of care (77 patients) or weekly subcutaneous injections of romiplostim (157 patients). Primary end points were incidences of treatment failure and splenectomy. Secondary end points included the rate of a platelet response (a platelet count >50×10 9 per liter at any scheduled visit), safety outcomes, and the quality of life. Results The rate of a platelet response in the romiplostim group was 2.3 times that in the standard-of-care group (95% confidence interval [CI], 2.0 to 2.6; P

Summary. Background: p45 NF-E2 is a bZIP transcription factor crucial for thrombopoiesis, as indicated by the fact that loss of p45 NF-E2 function results in dramatic embryonic lethal thrombopoietic defects and its overexpression boosts platelet release. Objectives: In the present study, we set out to identify the sequences responsible for p45 NF-E2 nuclear import, evaluate its transport mechanism and ascertain its functional significance. Methods: A series of p45 NF-E2 deletion constructs fused to green fluorescent protein (GFP) was created and their cellular localization examined in mammalian cells, with the factor responsible for nuclear import identified using an in vitro transport assay. A p45 NF-E2 derivative mutated in the nuclear targeting sequence (NLS) was generated and its biological activity compared with wild type (wt) in luciferase assays, and proplatelet and platelet production measured in murine megakaryocytes transduced with a retroviral vector. Results: Here we show that residues 271–273 are essential for nuclear import of p45 NF-E2 in COS-7 and in primary bone marrow cells. The p45 NF-E2 NLS facilitates nuclear import specifically via importin (IMP) 7. Although within the DNA-binding domain of p45 NF-E2, the NLS is not essential for DNA-binding, but is crucial for transcriptional activation and biological activity; where, in contrast to wt, a mutant derivative with a mutated NLS failed to promote proplatelet and platelet production in murine megakaryocytes. Conclusions: The NLS is critical for p45 NF-E2 function, with the present study being the first to demonstrate the importance of NLS-dependent nuclear import of p45 NF-E2 for platelet development.

Expression of the chemokine receptor CXCR4 by haematopoietic stem cells (HSCs) is believed to influence the process of these cells 'homing' back to the bone marrow post-transplantation, in response to the stromal cell-derived factor-1 gradient, followed by engraftment. The primary aim of this retrospective study was to compare reinfused CD34(+) cell dose, assessed from the fresh collection, with the post-thaw viable (v) CD34(+) and vCD34/CXCR4(+) dual positive cell dose as predictors of haematopoietic recovery in multiple myeloma patients undergoing autologous stem cell transplantation. Cryopreserved samples from stem cell collections of 27 myeloma patients were analysed for CD34 and CXCR4 expression and times to haematological engraftment measured. Dosage of transplanted vCD34(+) cells was on average 79% of the original calculation from the fresh collection bag (range 29-98%). The median percentage of vCD34+ cells co-expressing CXCR4 was 37% (3.7-97%). Surface expression of CXCR4 by thawed vCD34(+) cells was closely correlated to complementary DNA levels. The median dose of CD34/CXCR4(+) cells in the autografts was 1.2 × 10(6)/kg (0.2-3.0 × 10(6)/kg) compared with 3.3 × 10(6)/kg for transplanted vCD34(+) cells (1.2-5.5 × 10(6)/kg). Both CD34 and vCD34 doses correlated with neutrophil engraftment (p 0.005) although vCD34/CXCR4(+) dose did not. However, patients given a higher dose of CD34/CXCR4(+) cells (≥1.75 × 10(6)/kg) showed a faster time to platelet recovery (p 0.05) than those given a lower dose (≤0.42 × 10(6)/kg). These results warrant further study of CD34/CXCR4 expression by mobilised HSCs and the relationship to platelet recovery post-transplantation on a larger cohort of patients.

Background Platelet-derived growth factor is involved in the regulation of hematopoiesis. Imatinib mesylate, a platelet-derived growth factor receptor inhibitor, induces thrombocytopenia in a significant proportion of patients with chronic myeloid leukemia. Although our previous studies showed that platelet-derived growth factor enhances megakaryocytopoiesis in vitro, the in vivo effect of platelet-derived growth factor in a model of radiation-induced thrombocytopenia has not been reported. Design and Methods In this study, we investigated the effect of platelet-derived growth factor on hematopoietic stem/progenitor cells and platelet production using an irradiated-mouse model. We also explored the potential molecular mechanisms of platelet-derived growth factor on thrombopoiesis in M-07e cells. Results Platelet-derived growth factor, like thrombopoietin, significantly promoted the recovery of platelets and the formation of bone marrow colony-forming unit-megakaryocyte in irradiated mice. Histology confirmed the protective effect of platelet-derived growth factor, as shown by an increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, platelet-derived growth factor had a similar anti-apoptotic effect as thrombopoietin on megakaryocytes. We also demonstrated that platelet-derived growth factor activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Conclusions Our findings show that platelet-derived growth factor enhances platelet recovery in mice with radiation-induced thrombocytopenia. This radioprotective effect is likely to be mediated via platelet-derived growth factor receptors with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockage of platelet-derived growth factor receptors may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. © 2010 Ferrata Storti Foundation., link_to_OA_fulltext

The microstructure and rheological properties of thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) microgels cross-linked with methylenebis-acrylamide (BA) were examined by dynamic light scattering and rheological techniques. As the temperature was increased from 10 to 50 °C, the particles diameter decreased by approximately two times near the volume phase transition temperature, Tv of between 30 and 35 °C. The addition of salt to the microgel dispersion provides competition for the water molecules hydrating the PNIPAM chains thus weakening the PNIPAM–H2O hydrogen bonds and the microgel progressively deswelled. The validity and limitation of the semi-empirical approach to model charged soft microgel particles developed previously were tested on this thermo-responsive system. A variable specific volume, k was introduced to convert the mass concentration to effective volume fraction. With increasing concentration, inter-particle repulsive force was enhanced, which overcame the osmotic force inside the soft particle, resulting in the expulsion of solvent from the swollen particles, and the particle shrank. The viscosity data for PNIPAM microgels at varying solution temperatures and ionic strength showed excellent agreement with the modified Krieger–Dougherty (K–D) model.

A smart and recyclable oil absorber based on pH-responsive block copolymer modified magnetic nanoparticles is constructed for effective separation of oil-in-water emulsions. Superparamagnetic iron(III) oxide (Fe3 O4 ) nanoparticles are designed as the core materials for providing the separation force, and surface modification with poly(4-vinylpyridine-b-dimethyl siloxane-b-4-vinylpyridine) (P4VP-PDMS-P4VP) block copolymer is performed to supply switchable oil wettability. The mean hydrodynamic radius of the nanoparticles increases from 43.8 ± 3.6 to 75.2 ± 5.3 nm when the neutral pH is reduced to 3, due to the protonation and subsequent swelling of P4VP segments at lower pH. The nanoparticles show excellent separation performance and can absorb octadecene up to 78.2 times of their own weight, simply by switching the pH of the oil-in-water emulsion treated with the nanoparticles and using a magnetic field. The different dipole of P4VP-PDMS-P4VP and protonated P4VP-PDMS-P4VP, as confirmed by density functional theory calculations, is responsible for the different oil miscibility and wettability of the nanoparticles.

Plant Preservative Mixture (PPMTM), a proprietary mixture of two broad-spectrum isothiazolone biocides, was recently introduced as a prophylactic anti-bacterial agent in plant tissue culture. Its efficacy for eradicating endophytic Sphingomonas paucimobilis in Petunia hybrida was tested. The presence of bacterial cells in intercellular spaces of parenchyma tissues in the cortex and pith, in the lumen of xylem vessels and within sclerenchyma cells, could be detected in histo-sections by both light and transmission electron microscopy. The eradication protocol involved infiltration of infected axillary buds with PPM solution under vacuum. Quantitation by high performance liquid chromatography (HPLC) revealed that axillary buds from upper young nodes absorbed significantly more PPM compared with those from the lower nodes. Indexation indicated successful eradication of bacterial contaminants from upper young axillary buds after vacuum-infiltration with 5 ml l−1 PPM.

Previously published guidelines for the diagnosis and management of primary immune thrombocytopenia (ITP) require updating largely due to the introduction of new classes of therapeutic agents, and a greater understanding of the disease pathophysiology. However, treatment-related decisions still remain principally dependent on clinical expertise or patient preference rather than high-quality clinical trial evidence. This consensus document aims to report on new data and provide consensus-based recommendations relating to diagnosis and treatment of ITP in adults, in children, and during pregnancy. The inclusion of summary tables within this document, supported by information tables in the online appendices, is intended to aid in clinical decision making.

Well-defined nanostructured surface with domains of dimension ∼20 nm was formed by the self-assembly of brush-type amphiphilic block copolymers of poly[poly(ethylene glycol)methyl ether methacrylate]-block-poly(2,3,4,5,6-pentafluorostyrene) (P(PEGMA)-b-PPFS) which demonstrate promise in discouraging barnacle settlement as proven using laboratory settlement assays and marine field tests.

Background Side population cells are a rare subset of cells found in the adult heart that are highly enriched for stem and progenitor cell activity. Recent studies have suggested that Sca1+/CD31− cardiac side population cells are capable of differentiation into cardiomyocytes in vitro . However, the response of these cells to myocardial injury remains unknown in vivo . Methods Sca1+/CD31− cardiac side population cells were isolated from mouse (C57BL6/J) hearts by FACS. These cells were labeled and delivered via an intramyocardial injection into an infracted mouse heart. The differentiation potential of these cells was determined by immunohistochemistry two weeks later. We further tested the migration potential and the relationship of SDF-1α/CXCR4 to these cells. Results The transplanted cells were found to express cardiomyocyte or endothelial cell specific markers. Furthermore, when these cells were transplanted into non-infarct myocardium after myocardial infarction, they were found in the damaged myocardium. Consistent with their homing property, we found that SDF-1α and CXCR4 were up-regulated in the damaged myocardium and on Sca1+/CD31− cardiac side population cells respectively following myocardial infarction. We also show that SDF-1α induced migration of Sca1+/CD31− cardiac side population cells in vitro . Conclusions Our results have suggested that Sca1+/CD31− cardiac side population cells are able to migrate into damaged myocardium from non-ischemic area of the heart and differentiate into both cardiomyocyte- and endothelial-like cells following acute ischemic injury. The SDF-1α/CXCR4 system might play an important role in the migration of these cells.

Thrombocytopenia following heparin administration can be associated with an immune reaction, now referred to as heparin-induced thrombocytopenia . HIT is essentially a prothrombotic disorder mediated by an IgG antiplatelet factor 4/heparin antibody, which induces platelet, endothelial cell, monocyte, and other cellular activation, leading to thrombin generation and thrombotic complications. Indeed, HIT can also be regarded as a serious adverse drug effect. Importantly, HIT can be a life-threatening and limb-threatening condition frequently associated with characteristically severe and extensive thromboembolism rather than with bleeding. This article provides an overview of HIT, with an emphasis on the clinical diagnosis and management.

Five experiments were implemented to collect information related to the effects of fluazifop-p-butyl (active chemical in grass selective herbicides, Fusilade® and Fusilade Forte™) on seed germination, seedling emergence, growth and health of species native to southwest Australia (a grass and non-grasses), together with several co-occurring introduced species (grasses and a non-grass). Experiments investigated effects of herbicide concentrations, seed burial depths, seed-sowing times since herbicide application and application locations (foliage versus soil). Both herbicides, at half to quadruple strength of recommended field application concentrations, adversely affected development of native and introduced species, both grasses and non-grasses. Herbicidal effects were observed during the seed germination phase, and if germination had occurred, during seedling emergence and, finally, during plant establishment. However, effects were more pronounced after seed germination, particularly on development of seedlings and plants, with retardation and/or discoloration of either radicles or shoots. Not unexpectedly, seedlings from seeds buried deeper in the sand medium (20 mm) struggled to emerge. Both herbicides demonstrated residual characteristics by impeding seedling emergence and growth from seeds sown at various dates (up to maximum test duration of 3 weeks) following exposure of the sand medium to the herbicides. Further, herbicide application to sand only, produced effects on 5-6 months old plants that were similar as application to foliage only, demonstrating herbicide uptake from sand. While the findings support independent research, they contradict the purported herbicide characteristics by commercial sources - grass selective, post-emergent, non-residual, rapid breakdown and active through foliar application only. Implications of these herbicides for biodiversity conservation are discussed.

Diagnosis and management of immune thrombocytopenic purpura (ITP) remain largely dependent on clinical expertise and observations more than on evidence derived from clinical trials of high scientific quality. One major obstacle to the implementation of such studies and in producing reliable meta-analyses of existing data is a lack of consensus on standardized critical definitions, outcome criteria, and terminology. Moreover, the demand for comparative clinical trials has dramatically increased since the introduction of new classes of therapeutic agents, such as thrombopoietin receptor agonists, and innovative treatment modalities, such as anti-CD 20 antibodies. To overcome the present heterogeneity, an International Working Group of recognized expert clinicians convened a 2-day structured meeting (the Vicenza Consensus Conference) to define standard terminology and definitions for primary ITP and its different phases and criteria for the grading of severity, and clinically meaningful outcomes and response. These consensus criteria and definitions could be used by investigational clinical trials or cohort studies. Adoption of these recommendations would serve to improve communication among investigators, to enhance comparability among clinical trials, to facilitate meta-analyses and development of therapeutic guidelines, and to provide a standardized framework for regulatory agencies.

Objective NF-E2 is a prime regulator of megakaryocyte (MK) terminal differentiation and platelet release. By overexpressing the p45 subunit of NF-E2, we aim to increase the proportion of mature MKs and the potential for platelet production in vitro and in vivo. Methods Retroviral vectors expressing p45-NF-E2 together with the enhanced green fluorescent protein (eGFP) were used to transduce murine bone marrow cells (BMCs). Aspects of MK differentiation, proliferation, proplatelet, and platelet production were evaluated. Results Compared to controls, a higher proportion of BMCs overexpressing p45-NF-E2 were found to express the MK markers CD41, CD42a, and CD42b, with some effect on cell proliferation. Early MK differentiation, characterized by colony-forming unit (CFU)-MK formation, was enhanced by p45-NF-E2 overexpression at the expense of CFU-granulocyte macrophage development. An increased number of acetylcholinesterase + MKs was also observed in NF-E2 ++ cultures. Although endomitosis was found not to be affected, the resultant upregulation of NF-E2 target genes was also followed by significant increases in proplatelet and functional platelet production. Transplantation of enriched MK progenitor cells overexpressing p45-NF-E2 into lethally irradiated mice resulted in a threefold increase in eGFP + /NF-E2 ++ platelet production in vivo over 10 days, although no appreciable expansion in their number was observed over 32 days. Conclusion These results suggest that enforced expression of p45-NF-E2 selectively enhances many aspects of MK differentiation, including MK maturation, proplatelet formation, and platelet release. In addition, p45 overexpression increases MK commitment during early megakaryopoiesis, while inhibiting white blood cell differentiation.

Hematological changes in patients with Severe Acute Respiratory Syndrome (SARS) are common and frequently include thrombocytopenia. Using a ELISA method, we found an increase in thrombopoietin (TPO) levels in the plasma of convalesced SARS patients (290 ± 53 pg/ml) and active SARS patients (251 ± 23 pg/ml) comparing to that from normal control patients (228 ± 17 pg/ml). In addition, the plasma from active SARS patients had an inhibitory effect on CFU-MK formation, which could be neutralized by anti-TGF-β antibodies. In the experiment to determine whether SARS-CoV can directly infect hematopoietic stem cells and megakaryocytic cells, incubation of the cells with SARS-CoV did not show active infection. Our findings of increased TPO levels in the plasma of SARS patients provide a possible explanation for the genesis of thrombocytosis, which frequently develops from thrombocytopenia in SARS patients.

This review presents an overview on the research on pH-responsive microgel particles in the last 10 years. Microgels are cross-linked latex particles that are swollen in a good solvent. Significant quantitative studies have been conducted to investigate the swelling behavior (microscopic) and rheological (macroscopic) properties of the pH-responsive microgel particles as a function of neutralization degree, ionic strength, and cross-linked density. Mono-dispersed, alkali-swellable microgels containing carboxylic acid lattices, whose properties display extreme pH sensitivity in water is considered in detail in terms of swelling behavior and rheological properties. Their stability in solution and ability to undergo reversible volume phase transitions in response to pH makes them ideal model systems for the development of a semi-empirical as well as theoretical approach for predicting the viscosity of dilute and concentrated hard and soft sphere systems. The review concludes with a discussion of some recent applications of pH-responsive microgel particles.

Viscometry, static light scattering and turbidimetry studies were performed to investigate the interaction between pH-responsive microgels consisting of methacrylic acid (MAA) and ethyl acrylate (EA) copolymers cross-linked with di-allyl phthalate (DAP) and the solvent environment. The characteristics of microgel particles and their corresponding interaction potential were manipulated by varying the following: (a) degree of neutralization of MAA groups; α (b) salt concentrations and (c) microgel cross-linked density. Three independent interaction parameters, i.e. Huggins constant, Kh, second virial coefficient, A2 and apparent second virial coefficient, Bapp, which describe the degree of particle–solvent interactions, were obtained from these techniques. A scaling relationship between the three parameters given as: Bapp/Kh∝αM¯wA2 (where M¯w represents the weight-average molecular weight), was proposed. Good agreement was observed, where all the data for the three different salt concentrations of each microgel with different cross-linked densities collapsed onto a single master curve. This suggested that all three techniques can accurately predict the degree of interaction between microgel particle and solvent environment. Turbidimetry, being the simplest among the three techniques is the most convenient method.

Background Evidence-based guidelines recommend that acutely ill hospitalized medical patients who are at risk of venous thromboembolism (VTE) should receive prophylaxis. Our aim was to characterize the clinical practices for VTE prophylaxis in acutely ill hospitalized medical patients enrolled in the International Medical Prevention Registry on Venous Thromboembolism (IMPROVE). Methods IMPROVE is an ongoing, multinational, observational study. Participating hospitals enroll the first 10 consecutive eligible acutely ill medical patients each month. Patient management is determined by the treating physicians. An analysis of data on VTE prophylaxis practices is presented. Results From July 2002 to September 30, 2006, 15,156 patients were enrolled from 52 hospitals in 12 countries, of whom 50% received in-hospital pharmacologic and/or mechanical VTE prophylaxis. In the United States and other participating countries, 52% and 43% of patients, respectively, should have received prophylaxis according to guideline recommendations from the American College of Chest Physicians (ACCP). Only approximately 60% of patients who either met the ACCP criteria for requiring prophylaxis or were eligible for enrollment in randomized clinical trials that have shown the benefits of pharmacologic prophylaxis actually received prophylaxis. Practices varied considerably. Intermittent pneumatic compression was the most common form of medical prophylaxis utilized in the United States, although it was used very rarely in other countries (22% vs 0.2%, respectively). Unfractionated heparin was the most frequent pharmacologic approach used in the United States (21% of patients), with low-molecular-weight heparin used most frequently in other participating countries (40%). There was also variable use of elastic stockings in the United States and other participating countries (3% vs 7%, respectively). Conclusions Our data suggest that physicians' practices for providing VTE prophylaxis to acutely ill hospitalized medical patients are suboptimal and highlight the need for improved implementation of existing evidence-based guidelines in hospitals.

Nucleocytoplasmic transport of GATA-4 is important in maintaining and regulating normal cardiogenesis and heart function. This report investigates the detailed mechanisms of GATA-4 nuclear transport. We characterized a nonclassical nuclear localization signal between amino acids 270 and 324 that actively transports GATA-4 into the nucleus of both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial arginine residues within this region that mediate active transport predominately through the nonclassical pathway via interaction with importin β. These four residues were also essential for the DNA binding activity of GATA-4 and transcriptional activation of cardiac-specific genes. Interestingly, mutation of these residues not only inhibited DNA binding and gene transcription but also resulted in a preferential accumulation of the GATA-4 protein in distinct subnuclear speckles. A cardiac myocyte-specific, chromosome maintenance region 1-dependent nuclear export signal consisting of three essential leucine residues was also identified. The current study provides detailed information on the nuclear shuttling pathways of GATA-4 that represents an additional mechanism of gene regulation.

Emulsion copolymerization of poly(methacrylic acid) and poly(2-(diethylamino)ethyl methacrylate) (PMAA/PDEA) yielded pH-responsive polyampholyte microgels of 200-300 nm in diameter. These microgels showed enhanced hydrophilic behavior in aqueous medium at low and high pH, but formed large aggregates of approximately 2500 nm at intermediate pH. To achieve colloidal stability at intermediate pH, a second batch of microgels of identical monomer composition were synthesized, where monomethoxy-capped poly(ethylene glycol)methacrylate (PEGMA) was grafted onto the surface of these particles. Dynamic light-scattering measurements showed that the hydrodynamic radius, Rh, of sterically stabilized microgels was approximately 100 nm at intermediate pH and increased to 120 and 200 nm at pH 2 and 10, respectively. Between pH 4 and 6, these microgels possessed mobility close to zero and a negative second virial coefficient, A2, due to overall charge neutralization near the isoelectric pH. From the Rh, mobility, and A2, cross-linked MAA-DEA microgels with and without PEGMA retained their polyampholytic properties in solution. By varying the composition of MAA and DEA in the microgel, it is possible to vary the isoelectric point of the colloidal particles. These new microgels are being explored for use in the delivery of DNA and proteins.

A series of pH-responsive amphiphilic poly(N,N-dimethylaminoethyl methacrylate)-block-poly(D-lactic acid)-block-poly(N,N-dimethylaminoethyl methacrylate) conjugated with poly(ethylene glycol) (D-PDLA-D@PEG) and D-PLLA-D@PEG copolymers were synthesized using a combination of ring-opening polymerization and atom-transfer radical polymerization followed by sequential quaternization of PDMAEMA chains and azide-alkyne click reaction with alkyne-end PEG. Gel permeation chromatography, nuclear magnetic resonance, and Fourier transform infrared spectroscopy results demonstrate the successful synthesis of the copolymers, and the conjugated PEG percentages in the copolymers can be tuned by the feeding ratios in the quaternization reaction. Conjugating PEG onto the PDMAEMA segments also successfully facilitated the D-PDLA-D@PEG, D-PLLA-D@PEG, and its corresponding 1:1 D/L mixtures to be dissolved directly in aqueous solution at the desired concentration range without using any organic solvents unlike the copolymers without PEG conjugation (D-PDLA-D and D-PLLA-D). We demonstrate control over micellar size, charge, and stability via three different preparation pathways, i.e., solution pH, percentages of PEG conjugation in the copolymers, and formation of PLA stereocomplex in micellar core. Static and dynamic light scattering studies demonstrate that the size of the core-shell micelles increases when the solution pH is reduced due to the protonation of the PDMAEMA segments that caused the osmotic pressure within the micelle to increase until the micelles reached a maximum size. It is interesting to note that the micelles formed by 1:1 D/L mixtures have larger swelling ratios as well as aggregation number and hydrodynamic radius that do not change significantly with pH and dilution, respectively, as compared to micelles formed from individual D or L forms of the copolymers. The enhanced stability of the pH-responsive micelles prepared by direct dissolution of the 1:1 D/L mixtures of the PEG conjugated PLA-based polyelectrolytes in aqueous medium is attributed to the stereocomplex formation between PLLA and PDLA in the micellar core as confirmed by wide-angle X-ray scattering measurements.

Interleukin-16 (IL-16) induces the chemotaxis and activation of mast cells (MCs) and other cell types. While it has been concluded that CD4 is the primary IL-16 receptor on T cells, at least one other IL-16 receptor exists. We now show that the IL-16–responsive human MC line HMC-1 lacks CD4, and that the IL-16–mediated chemotactic and Ca2+ mobilization responses of this cell can be blocked by anti-CD9 monoclonal antibodies (mAbs) but not by mAbs directed against CD4 or other tetraspanins. Anti-CD9 mAbs also inhibited the IL-16–mediated activation of nontransformed human cord blood–derived MCs and mouse bone marrow–derived MCs by 50% to 60%. The chemotactic response of HMC-1 cells to IL-16, as well as the binding of the cytokine to the cell's plasma membrane, was inhibited by CD9-specific antisense oligonucleotides. CD9 is therefore essential for the IL-16–mediated chemotaxis and activation of the HMC-1 cell line. In support of this conclusion, IL-16 bound to CD9-expressing CHO cell transfectants. The ability of wortmannin and xestopongin C to inhibit the IL-16–mediated chemotactic response of these cells suggests that the cytokine activates a phosphatidylinositol 3-kinase (PI3K)/inositol trisphosphate–dependent signaling pathway in MCs. This is the first report of a tetraspanin that plays a prominent role in a cytokine-mediated chemotactic response of human MCs.

The microstructure and rheological properties of core–shell pH-responsive microgels consisting of poly(methyl methacrylate) (PMMA) particles grafted with a soft layer of methacrylic acid–ethyl acrylate (MAA–EA) cross-linked with di-allyl phthalate (DAP) were examined using dynamic light scattering and rheological techniques. The validity and limitation of the semi-empirical approach to model charged soft microgel particles developed by our group were tested on this core–shell system. The viscosity data for three different core–shell particles showed excellent agreement with the modified Krieger–Dougherty (K–D) model. Good agreement was also observed when our semi-empirical approach was compared against a theoretical model, which confirmed the validity of the semi-empirical approach to model charged soft particles. In addition, we confirmed that the new scaling law which relates the swelling ratio Q of microgels as a function of neutralization degree, α, average number of monomers between two cross-links, Nx, molar fraction of acidic units, y and concentration of mobile counter-ions, C K + and C Na + , represented as ( N x / c 0 ) ( C K + + C Na + ) Q + Q 2 / 3 is proportional to yNxα. All the core–shell data at varying ionic strength and mobile counter-ions concentrations fall onto a master curve.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets coated with mainly antibodies against platelet GPIIb/IIIa and GPIb/IX are destroyed in the spleen. Recent evidence suggests that platelets are also destroyed by cytotoxic T cells. The diagnosis is made by exclusion for other causes of thrombocytopenia. As routine blood counts are becoming more available, many mild cases of ITP (platelets30 x 10(9) L(-1)) are being diagnosed and they usually do not require treatment. In patients with platelet counts persistently30 x 10(9) L(-1), treatment with corticosteroids, and/or intravenous immunoglobulin (IVIG) or anti-D may be required. The primary goal of treatment is to maintain the platelet count at a safe level with minimal side effects. After 3-6 months, if spontaneous remission has not occurred and if the side effects are significant, splenectomy is recommended. This is the single most effective treatment of ITP. The refractory patients who fails splenectomy and subsequently first- and second-line therapies, is a management dilemma. Therapeutic options are limited, available treatments potentially toxic and the chances of sustained response low. Observation with no active treatment is a reasonable option. With the increased availability of the thrombopoietic agents in the future, there may be a good prospect of keeping the platelet counts of these refractory patients at a safe long-term level with one of these drugs.

A turbidimetric analysis of particle interaction of model pH-responsive microgel systems consisting of methacrylic acid-ethyl acrylate cross-linked with diallyl phthalate in colloidal suspensions is described. The structure factor at zero scattering angle, S(0), can be determined with good precision for wavelengths greater than 500 nm, and it measures the dispersion's resistance to particle compression. The structure factor of microgels at various cross-linked densities and ionic strengths falls onto a master curve when plotted against the effective volume fraction, phi(eff) = kc, which clearly suggests that particle interaction potential and osmotic compressibility is a function of effective volume fraction. In addition, the deviation of the structure factor, S(0), of our microgel systems with the structure factor of hard spheres, S(PY)(0), exhibits a maximum at phi(eff) approximately 0.2. Beyond this point the osmotic de-swelling force exceeds the osmotic pressure inside the soft particles resulting in particle shrinkage. Good agreement was obtained when the structural properties of our microgel systems obtained from turbidimetric analysis and rheology measurements were compared. Therefore, a simple turbidimetric analysis of these model pH-responsive microgel systems permits a quantitative evaluation of factors governing particle osmotic compressibility.

In this issue of Blood, Jaax and colleagues show that heparin-PF4 antibodies cross-reacted with nucleic acid (NA)–PF4 complexes and induced platelet activation, suggesting that NA-PF4 can potentially cause a heparin-induced thrombocytopenia (HIT)–like prothrombotic disorder.

The microstructure and rheological properties of swellable nano-particles consisting of cross-link methacrylic acid/ethyl acrylate were examined using dynamic light scattering (DLS) and rheological techniques. A unified semi-empirical approach for predicting the viscosity of dilute and concentrated systems was proposed, where a variable specific volume, k was introduced to convert the mass concentration to effective volume fraction. The viscosity data for four different microgel systems showed excellent agreement with the modified Krieger–Dougherty model. The parameters, c 0 and m for the expression, k − k min / k 0 − k min =[1+( c / c 0 ) 2 ] − m as a function of degree of neutralization ( α ) were determined. We observed that c 0 decreases with α , while m increases with α . The validity of the new approach was compared against the theoretical model for determining the effective volume fraction, φ=( Q /V m c 0 )(ρ s /ρ p )c and excellent agreement was observed.

We describe a unified semi-empirical approach for predicting the viscosity of dilute and concentrated hard and soft sphere systems. A variable specific volume, k was introduced to convert the mass concentration to effective volume fraction. With increasing particle concentration, the concentration of free counter-ions in the solution can be large enough to induce an osmotic de-swelling of soft particles, resulting in the particle shrinkage. The viscosity data for four different microgel systems at different neutralization degree showed excellent agreement with the modified Krieger–Dougherty model.

Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbalpha genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.

Thrombospondin-1 (TSP-1) is an inhibitor of angiogenesis, inducing apoptosis of the endothelial cells via CD36 signaling mechanism. We investigated CD36 expression and the effect of TSP-1 on megakaryocytopoiesis, with and without pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), and with and without blocking TSP-1 binding with receptor CD36 on megakaryocytic cells. Our data showed that TSP-1 induced a dose-dependent growth inhibition in both murine and human colony forming unit-megakaryocyte (CFU-MK) assays and significantly counteracted the mitogenic effect from PEG-rHuMGDF. Moreover, the growth suppression induced by TSP-1 was correlated with CD36 expression in megakaryocytic cell lines, where growth inhibition was demonstrated in CD36 positive (Meg-01, Dami and CHRF-288-11) but not in CD36 negative (M-07e) cell lines. More importantly, the inhibitory effect of TSP-1 on both human CFU-MK and Meg-01 cells was partially but significantly reversed by the addition of FA6-152 (anti-CD36), a blocking antibody which blocks the access of TSP-1 to CD36 receptor, suggesting that the TSP-1-induced inhibition of megakaryocytopoiesis is probably mediated in part by the binding of TSP-1 to CD36 expressed on the megakaryocytic progenitors. Thus, our findings represent the first demonstration that TSP-1 inhibits in vitro megakaryocytopoiesis via interaction with CD36.

Human platelet survival studies have been hampered due to the lack of a suitable animal model. Transfusion of human platelets into immune competent animals leads to the rapid destruction of these platelets by naturally occurring xenoantibodies. The NOD/SCID mouse lacks T and B cells and therefore lack natural antibodies that could destroy infused human platelets [1]. Because of this property, human platelets given to the mouse intravenously circulate for several days, permitting the model to be used for testing the ability of human antibodies to cause platelet destruction in vivo [2–7]. Preliminary studies have demonstrated the usefulness of NOD/SCID mouse model to monitor the survival and immune destruction of human platelets. However, differences exist between the research groups regarding the method of PLT injection, the amount and route of antibody injection and the preparation of blood samples collected from the animal making the results poorly comparable. Basically, in all laboratories resting human platelets are injected intravenously into the mouse circulation, where they can, in the absence of platelet-reactive antibodies, circulate up to 48h [8]. After estimating a baseline value (100%) of human platelets, platelet-reactive antibodies (with or without drug administration) can be infused. The impact of these antibodies on the survival of human platelets can then be analyzed by taking blood samples over time from the mouse [8]. Methodological details that require attention in this model include: platelet preparation and resuspension in plasma or ‘synthetic plasma’, the concentrations and volume of applied analytes (platelet, antibody or drug), the route of platelet injection (retro-orbital injection or tail vein injection) and antibody injection (intravenous, intra-peritoneal). The method of data capture, including time points of blood sampling and subsequent sample preparation for analysis, percentage of circulating human platelets and software details should also be reported in detail. Additional steps required for answering the scientific questions, for example platelet preincubation with a drug of interest or an antibody in pooled plasma or ‘synthetic plasma’, should also be reported [2, 9]. Surprisingly, application procedures and the amount of injected platelets and antibodies have only been loosely defined and standardization should be carried out in order to improve the reproducibility of the procedures and to enable reliable comparison of the results. This report is not didactic in relation to how to measure the survival of human platelets using the NOD/SCID mouse model. Its purpose is to suggest standardized procedures and define variables that should be considered when presenting methodology in published reports. The presented procedures were introduced and discussed during the meetings of the Subcommittee of Platelet Immunology of the Scientific and Standardization Committee Liverpool 2012 and Milwaukee 2014. Suggestions were introduced to the SSC members and the presented recommendations had unanimous agreement. Adopting these recommendations will be of advantage for investigators and laboratories to reduce imprecision and harmonize results and will allow other laboratories to readily reproduce reported methods and findings and interpret results appropriately.

Primary immune thrombocytopenia is an autoimmune disease mediated by antiplatelet autoantibodies that cause platelet destruction and suppression of platelet production. In vitro effects of autoantibodies on megakaryocyte production and maturation have been reported recently. However, the impact of these autoantibodies on crucial megakaryocyte functions, proplatelet formation and subsequent platelet release, has not been evaluated. We examined the effects of serum and IgG from 19 patients with immune thrombocytopenia using day 8 or 9 megakaryocytes (66.3 ± 10.6% CD41(+)), derived from cord blood hematopoietic stem cells (CD34(+)). The number of proplatelet-bearing megakaryocytes, the number of platelets released in the culture, total megakaryocyte numbers, ploidy pattern and caspase activation were measured at various times after treatment. After 5 days of treatment the number of proplatelet-bearing megakaryocytes was significantly decreased by 13 immune thrombocytopenia autoantibodies relative to the control group (P

The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of >2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

Background: Primary care chronic kidney disease (CKD) registers report widely varying prevalence within the UK. We examined the effects of laboratory ascertainment and adjusting for practice-level variables on the variation in CKD prevalence. We carried out an Ayrshire-wide laboratory database analysis of primary care practices (PCPs). Methods: We analysed 54 PCPs with 313 639 registered patients aged ≥18. All patients with a low estimated glomerular filtration rate (

To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbα polypeptide chain have been identified in these patients—a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbα chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbα, thereby influencing the stability of the GPIbα–vWf interaction.

Objective: The infusion of bone marrow cells into the damaged brain has been proposed as a new clinical practice for this disorder. Alternatively, hematopoietic growth factors may have a direct role on neural protection or have a mobilizing effect on bone marrow stem/progenitor cells to circulation for brain repair. Based on our previous findings, there are many similarities between megakaryocytes and neurons on functions and antigen expression such as neural marker MAP2, GFAP and Tau, 5-HT2A, 2B and 2C receptors (Stem Cells, 2014). Thrombopoietin (TPO) is a growth factor for megakaryocytic lineage. We postulate that TPO may play a role on neural protection or regeneration. The effect of TPO on nervous system has not been well investigated. Methods: To validate this hypothesis, we investigated the expression and role of TPO/TPO receptors in neural cells and a neonatal rat model of hypoxic-ischemic (HIE) brain damage. Results: To investigate the effect of TPO on in-vivo neural protection, a neonatal rat model of HIE brain damage was established. Brain injury was measured by the percentage weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significantly less brain atrophy in TPO treated animals (12.0±1.2% and 11.5±1.0%) when compared with the saline control (21.0±1.6% and 24.4±2.2%) at 7 and 28 days post-operation (P Conclusions: Our study provided the evidences showing the expression of TPO and TPO receptor (c-mpl) in neural cells and this effect may be mediated by c-mpl and Akt signaling. More importantly, our observation further demonstrated the functional role of TPO on neural protection in a rat model. These findings point to the possibility of a new strategy for treating brain damage by hematopoietic growth factors. Disclosures Yang: National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).