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Summary Cardiovascular disease, including myocardial infarction (MI), is the leading cause of death globally. Current antithrombotic medications used during MI treatment are predominantly directed towards platelet inhibition and, to a lesser extent, anticoagulation. Bleeding is a major risk of such treatment and could be circumvented by targeting other causative factors essential for arterial thrombus formation. We sought to re-evaluate the cellular composition of arterial thrombus in order to better understand mechanisms that lead to coronary artery thrombosis in acute MI. We performed detailed histological and immunohistochemical analysis of coronary artery thrombi aspirated from 26 patients undergoing emergency percutaneous coronary intervention for acute ST elevated myocardial infarction (STEMI). Coronary arterial thrombi had an unanticipated cellular heterogeneity. Neutrophil extracellular traps (NETs) were observed in thrombi as identified by anti-citrullinated histone 3 and anti-myeloperoxidase staining. Increased abundance of NETs was seen directly surrounding erythrocytes. Extracellular iron and erythrocyte fragments were also associated with areas of NETs suggesting a possible link. Our results shed light on potential involvement of erythrocytes in coronary arterial thrombosis through activation of platelets and induction of NETs. If supported by further in vitro and in vivo studies, novel therapies to inhibit NET formation or coagulation activation by erythrocyte release products, could bolster current myocardial infarction treatment.

In the effort to overcome the shortcomings such as brittleness and poor mechanical stability, and increase the competitive edge of renewable poly(lactic acid) PLA over conventional petroleum-based thermoplastics, silica rubber core-shell nanoparticles for effective PLA toughening were successfully synthesized by sequential ring opening polymerization (ROP). The core-shell structure was designed with silica as inner core, P(CL-mLA) as ‘rubber’ middle layer and terminal PDLA chains (SiO2-r-PDLA), to facilitate the stereocomplex formation with PLLA matrix for enhanced interface control. The PLLA/SiO2-r-PDLA nanocomposites were fabricated through solution blending-injection molding process. Nuclear magnetic resonance (1H NMR and 13C NMR) results confirmed the presence of grafted ’rubber’ and PDLA chains from the surface of silica particle. In addition, PLLA/SiO2-r-PDLA nanocomposites showed tremendous improvement in thermal and mechanical properties using differential scanning calorimetry (DSC) and tensile testing, respectively. Besides the formation of stereocomplex in the nanocomposites, a detailed study on the melt stability of these stereocomplex nanocomposites revealed a ‘memorized’ stereocomplex behavior, i.e., having the ability to perfectly reassemble after re-crystallization from melt (melt memory effect), when rubber segment is present. Finally, structure-deformation mechanisms were studied using scanning electron microscopy (SEM) and small angle x-ray scattering (SAXS). From SAXS, crazing was clearly observed whereas SEM revealed fibrillated structures. Thus, crazing and fibrillation are the key deformation mechanisms in PLLA/SiO2-r-PDLA system. The exciting new fillers could open up new horizons for PLA advanced composites applications.

An insightful understanding of the isothermal cold crystallization, melting behavior, and kinetics of the novel PHB-rubber copolymer ( PHB-di-rub )-reinforced polylactide (PLA) were determined. Incorporating 10 wt% PHB-di-rub significantly reduces the crystallization half-times (t1/2) by 32-fold, lowers the kinetic parameter (Kg) and folding surface energy (σe ), which show the rapid polymer chains folding and the super-nucleating capability due to simultaneous instigated nucleation and plasticization. Concurrently, the shear-thinning behavior verified the plasticization effect and the convergence of diffraction peak show development of primary α′-crystalline form that contributes to the PLAs' ultra-extensibility. This evocative finding could comprehend a valuable framework for the future development of high-performance PLA/PHB-based copolymer biocomposites with total green physiognomies.

Β2-Glycoprotein I (β2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of β2GPI was examined.The effects of nitrated (n)β2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. β2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. β2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated β2GPI loses this binding (p

Poly(lactic acid) or poly(lactide) (PLA) is a biodegradable and biocompatible thermoplastic polymer, being derived from renewable resources such as corn and sugar cane. The building block of PLA, lactic acid is chiral and the polymerization of lactic acids (or lactides) leads to isotatic, syndiotatic and atactic/heterotactic PLA primary structures. The stereoselective interaction between two complementary enantiomeric PLLA and PDLA has led to enhanced physical properties such as mechanical properties, thermal resistance and hydrolytic stability compared with the parent polymers. Progress in controlled and/or living polymerization techniques combined with other new synthetic methodologies has facilitated the preparation of PLA-based copolymers with complex architectures such as diblock, triblock, multiblock, star-shape block, comb-shape block and various PLA-grafted structures. The utilization of stereocomplexation strategy to these newly developed copolymers has opened avenues to access a variety of new materials with unique characteristics, including novel chemical functionalities, bioactivities, and smart (responsive to external stimulus) properties tailored for specific applications. This review presents recent advancements in the synthesis of PLA-based block/graft copolymers having complex architectures, with emphasis on the enhanced material performances induced by PLA stereocomplex formation. The origin of the enhanced thermal mechanical property observed in PLA stereocomplex is first discussed. The strong interaction resulted from stereocomplexation in PLA based copolymers could be exploited not only for fabrication of advanced therapeutic delivery carriers and tissue engineering devices, but also for stabilizing colloidal systems in microparticles, micelles and hydrogels, that further broaden the applications of PLA that could not have been attained with single PLLA or its copolymers. The stereocomplexation could also be used to tailor the interface interactions between fillers and PLA matrix that lead to higher strength and toughness of PLA.

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12–14 weeks gestation, but was undetectable at 8–11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors.

We have identified a biologically active component, panaxadiol saponins component (PDS-C), from Chinese ginseng herb extract. Panaxadiol saponins component contains five ginsenoside monomers with total purity of 92.44%. In this study, the BALB/c mouse model with immune thrombocytopenia (ITP) was established by injection of antiplatelet antibody every other day for 5 total times; the peripheral blood platelet counts steadily decreased to 20%-30% of normal levels and remained decreased for about 10 days. The antiplatelet antibody was derived from the sera of guinea pigs immunized with the platelets of BALB/c mice. Mice with ITP were treated with PDS-C at a low, a moderate, or a high dose for 10 consecutive days. We observed that the peripheral blood platelet counts of ITP mice were significantly higher than that of ITP controls (untreated) after treatment of PDS-C in a dose-dependent manner. Treatment with PDS-C also increased the mature megakaryocytes in the bone marrow of treated ITP animals with a concomitant decease of immature megakaryocyte precursors. Furthermore, macrophage phagocytosis of exogenous erythrocytes in the intra-abdominal cavity of ITP mice was inhibited by PDS-C treatment, indicating that PDS-C also could modulate immune function and may possibly prevent phagocytosis of antibody-coated platelets. Altogether, our findings suggest that PDS-C may have a dual role, promoting proliferation and differentiation of megakaryocytes, as well as modulating immune function, and it may therefore be very helpful in the treatment of ITP.

Polycaprolactone (PCL) and its copolymers are a type of hydrophobic aliphatic polyester based on hydroxyalkanoic acids. They possess exceptional qualities: biocompatibility; FDA approval for clinical use; biodegradability by enzyme and hydrolysis under physiological conditions and low immunogenicity. These critical properties have facilitated their value as sutures, drug delivery vehicles and tissue engineering scaffolds in pharmaceutical and biomedical applications. However, the hydrophobicity of PCL and its copolymers remains a concern for further biological and biomedical applications. One promising approach is to design and synthesize well-controlled PCL-based amphiphilic block copolymers. This review summarizes recent advances in the synthesis and self-assembly of PCL-containing amphiphilic block copolymers and their bio-related applications including drug delivery and tissue engineering.

Introduction Heparin induced-thrombocytopenia (HIT) has been well recognized in Western countries. However, there are no data in the Thai population. We therefore investigated the prevalence of anti-platelet factor 4 (PF4)/heparin antibodies, HIT, and its thrombotic complications in Thai patients undergoing cardiac surgery using unfractionated heparin. Materials and methods Seventy-three consecutive patients were prospectively enrolled in this study. Blood samples before operation and week 1, week 2, and week 3 after operation were collected from each patient for HIT antibody screening by enzyme-linked immunosorbent assay using IgG antibody specific to the PF4/heparin complex. Positive samples were further analyzed by 14 C-serotonin release assay. Complete blood count was performed daily during the first week, then weekly for 3 weeks. Results No patient had detectable anti-PF4/heparin antibodies at baseline. Five patients sero-converted during the course of the study for anti-PF4/heparin IgG: 3 (4.1%) at week 1, 4 (5.5%) at week 2, and 5 (6.8%) at week 3 after surgery. However, none of these patients had anti-PF4/heparin antibodies that resulted in 14 C-serotonin release to be considered clinically significant antibodies. Post-operative thrombocytopenia after the operation was found in 35 patients (47.9%), but was not considered to be caused by HIT. Thromboembolic events occurred in 3 patients (4.1%) during follow up; however, none of these patients had positive PF4/heparin antibody tests. Conclusions Our study represents the first study to examine Thai patients exposed to heparin in the context of cardiac surgery. We found a lower prevalence of positive anti-PF4/heparin antibodies and clinical HIT than previously published studies.

Thrombocytopenia is caused by immune reactions elicited by diverse drugs in clinical practice. The activity of the drug-dependent antibodies produces a marked decrease in blood platelets and a risk of serious bleeding. Understanding of the cellular mechanisms that drive drug-induced thrombocytopenia has advanced recently but there is still a need for improved laboratory tests and treatment options. This article provides an overview of the different types of drug-induced thrombocytopenia, discusses potential pathologic mechanisms, and considers diagnostic methods and treatment options.

Background Acutely ill hospitalized medical patients are at risk for VTE. We assessed the incidence of VTE in the observational International Medical Prevention Registry on Venous Thromboembolism (IMPROVE) study and derived VTE risk assessment scores at admission and associative VTE scores during hospitalization. Methods Data from 15,156 medical patients were analyzed to determine the cumulative incidence of clinically observed VTE over 3 months after admission. Multiple regression analysis identified factors associated with VTE risk. Results Of the 184 patients who developed symptomatic VTE, 76 had pulmonary embolism, and 67 had lower-extremity DVT. Cumulative VTE incidence was 1.0%; 45% of events occurred after discharge. Factors independently associated with VTE were previous VTE, known thrombophilia, cancer, age > 60 years, lower-limb paralysis, immobilization ≥ 7 days, and admission to an ICU or coronary care unit (first four were available at admission). Points were assigned to each factor identified to give a total risk score for each patient. At admission, 67% of patients had a score ≥ 1. During hospitalization, 31% had a score ≥ 2; for a score of 2 or 3, observed VTE risk was 1.5% vs 5.7% for a score ≥ 4. Observed and predicted rates were similar for both models (C statistic, 0.65 and 0.69, respectively). During hospitalization, a score ≥ 2 was associated with higher overall and VTE-related mortality. Conclusions Weighted VTE risk scores derived from four clinical risk factors at hospital admission can predict VTE risk in acutely ill hospitalized medical patients. Scores derived from seven clinical factors during hospitalization may help us to further understand symptomatic VTE risk. These scores require external validation.

A three amino acid sequence, Ser/Thr-Pro-Ser/Thr, was recently identified and characterized as a novel nuclear localization signal (Chuderland et al., 2008). The immediate-early gene product, early growth response-1 is a three zinc finger containing transcription factor implicated in a wide variety of pathologies, and has a bipartite nuclear localization domain identified two decades ago. Efficient nuclear localization of Egr-1 is vital to its function as a transcription factor. Interestingly, Egr-1 also contains a C-terminal SPS domain (residues 482-484 in murine Egr-1). We hypothesized that (482)SPS(484) may also serve as a novel nuclear localization signal in Egr-1. We found that this sequence directs Egr-1 to the nucleus in transfected Chinese hamster ovary cells and show by co-immunoprecipitation analysis that Egr-1 forms a complex with importin-7. (482)SPS(484) is required for Egr-1's interaction with importin-7. Moreover, importin-7 knockdown with RNAi showed that Egr-1 nuclear translocation is importin-7-dependent. This study demonstrates that the nuclear translocation of Egr-1 is partially dependent on (482)SPS(484) and involves importin-7, and sheds light on the molecular mechanisms regulating the cellular localization of this pathophysiologically important transcription factor.

Background Acutely ill, hospitalized medical patients are at risk of VTE. Despite guidelines for VTE prevention, prophylaxis use in these patients is still poor, possibly because of fear of bleeding risk. We used data from the International Medical Prevention Registry on Venous Thromboembolism (IMPROVE) to assess in-hospital bleeding incidence and to identify risk factors at admission associated with in-hospital bleeding risk in acutely ill medical patients. Methods IMPROVE is a multinational, observational study that enrolled 15,156 medical patients. The in-hospital bleeding incidence was estimated by Kaplan-Meier analysis. A multiple regression model analysis was performed to identify risk factors at admission associated with bleeding. Results The cumulative incidence of major and nonmajor in-hospital bleeding within 14 days of admission was 3.2%. Active gastroduodenal ulcer (OR, 4.15; 95% CI, 2.21-7.77), prior bleeding (OR, 3.64; 95% CI, 2.21-5.99), and low platelet count (OR, 3.37; 95% CI, 1.84-6.18) were the strongest independent risk factors at admission for bleeding. Other bleeding risk factors were increased age, hepatic or renal failure, ICU stay, central venous catheter, rheumatic disease, cancer, and male sex. Using these bleeding risk factors, a risk score was developed to estimate bleeding risk. Conclusions We assessed the incidence of major and clinically relevant bleeding in a large population of hospitalized medical patients and identified risk factors at admission associated with in-hospital bleeding. This information may assist physicians in deciding whether to use mechanical or pharmacologic VTE prophylaxis.

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.

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.

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.

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.

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.

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.

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.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).

Previous studies suggested that the cross-reactivity rates of low molecular weight (LMW) heparins with the antibody in heparin-induced thrombocytopenia (HIT) are more than 80%, whilst that of the LMW heparinoid (Orgaran) is relatively low at about 10%. These earlier studies were limited either in the number of patients studied, or in investigating only a single drug. They were also inadequate due to non-standardisation of testing conditions. This study compares three LMW heparins (Fragmin, Clexane, Fraxiparin) and a heparinoid (Orgaran) in their cross-reactivity rates with the HIT antibody. The sera of 45 HIT patients were tested using platelet aggregometry under standardised conditions. The cross-reactivity rates are: 7% (Orgaran), 89% (Fragmin), 83% (Clexane) and 86% (Fraxiparin). Although there are no controlled trials to determine the in vivo cross-reactivity rates of LMW heparins and heparinoid, the low in vitro cross-reactivity rate of the latter favours its use in HIT. Nevertheless, the therapeutic use of these drugs in HIT should be preceded by exclusion of in vitro cross-reactivity.

We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.

Aims To review the presentation and outcome of primary immune thrombocytopenia (ITP) using recent international working group guidelines.1 Methods 422 possible ITP cases (1/1/2005-1/6/2010) were examined at St George Hospital: medical records, laboratory databases. Results 67 primary ITP (54 new diagnoses, 13 relapsed): median age 57.8 years, platelet count 34 × 109/L, females 58%. 48% initial platelet count 54 newly diagnosed primary ITP: 16 treatment free from presentation, median follow-up 13.7 months. 23 required steroids, median duration 53 days. Median time to second-line therapies 49 days. 11 of 17 required second-line within 3 months of presentation. 24 intial platelet count Discussion Patients not requiring treatment at presentation usually remain treatment free. Conversely, most patients requiring additional therapies begin second-line within 3 months of presentation.

Immune thrombocytopenia is a common adverse effect of many drugs including heparin, quinine, GPIIb/IIIa inhibitors, antibiotics, ranitidine and rifampicin. Quinine-induced thrombocytopenia (QIT) is often considered a prototype of drug-induced immune thrombocytopenia (DIT). In DIT, the thrombocytopenia is severe (platelet count 9 /L) and has an abrupt onset. Bleeding is common. In QIT, the anti-platelet antibodies have specificity for platelet membrane proteins (GPIb-IX, GPIIb/IIIa, and occasionally GP V or PECAM-1) of which GPIb-IX is the major autoantigen. The antibody binds to platelets and causes premature platelet clearance by the reticuloendothelial system. We have located the antibody epitopestoresidues 283–293ofGPIba 1 andresiduesR110 to Q115 of GPIX. 2 Interestingly the epitope of three other DIT antibodies, namely quinidine-, ranitidine- and rifampicin-induced antibodies, have been mapped to the same region on GPIX as that of the antibody in QIT. This is probably because this region of GPIX is hyperflexible and is able to bind many drugs with diverse chemical structures to form drug/antigen complexes. We recently showed that the QIT antibodies also bound megakar-yocytes (MKs) via GPIb-IX, caused MK apoptosis and inhibited proplatelet and platelet formation. 3 These data suggest that there is a previously unrecognised mechanism of thrombocytopenia in DIT, mediated by antibody-induced decrease in platelet production. The management of DIT is cessation of the offending drug. Other therapeutic measures include the administration of glucocorticos-teroid and/or intravenous immunoglobulin.

Background Most heparin induced thrombocytopenia syndrome (HIT) clinical trials involve therapeutics with little evidence of safety and efficacy in renal impairment: all direct thrombin inhibitors accumulate in renal failure; fondaparinux is renally eliminated and relatively contraindicated in renal failure. Aims To determine frequency of renal impairment in patients suspected of HIT. Method Demographics and serum creatinine levels were recorded for every adult patient with HIT screen performed (1/11/2008-1/6/ 2010) ( n = 204). All screening was performed by solid phase ELISA using PF4 IgG by GTi Diagnostics. Results Median age 79, males 61%. Median and average serum creatinine at the time of HIT screening 104 μmol/L and 176 ± 25 μmol/L, respectively ( p = 0.05). Twenty-one screens positive, four equivocal. Twenty-five ‘non-negative’ patients: mean estimated glomerular filtration rate (eGFR) 58 ± 18 mL/min/1.73 m 2 ( p = 0.05) (abbreviated Modification of Diet in Renal Disease method); 48% eGFR 2 ; 64% serum creatinine >90 μmol/L. One hundred and fourteen HITS screens ordered from critical care units: 13 positive screens; 62% eGFR 2 , 77% serum creatinine >90 μmol/L. Discussion Renal impairment is common in patients suspected of HIT. In pivotal HAT studies (1994-1997), only 97 of 366 (26%) serum creatinine >90 μmol/L but these patients had three-fold increased risk of bleeding while on lepirudin. Critically unwell patients have high frequency of renal impairment. Clinical trials in HIT need to refocus on patients with renal impairment.