Thrombalexins: Cell-Localized Inhibition of Thrombin and Its Effects in a Model of High-Risk Renal Transplantation.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin-1 [TLN-1]) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

Abdominal wall hernia and mental health: patients lived experiences and implications for patient care.

BACKGROUND: Abdominal wall hernia (AWH) affects mental health and mental health questions are frequently included within Patient-Reported Outcome Measures (PROMS) for this patient population. However, these questions have not been informed by the subjective lived experiences of mental health in AWH patients. This study is the first to qualitatively examine how AWH affects patients' mental health. METHODS: Fifteen patients were interviewed from a purposive sample of AWH patients until no new themes emerged. Interviews explored patient thoughts and experiences of AWH and mental health. Data were examined using Interpretative Phenomenological Analysis (IPA). RESULTS: Three key themes pertaining to mental health were identified: "psychological and emotional distress", "identity disruption" and "coping mechanisms and support systems". CONCLUSION: Our findings illustrate that AWH is a pathology that can have a significant detrimental impact on people's mental health. This impact has implications for patient care and can be treated and managed through better psychological support. This support may positively affect AWH patient's experience and outcomes in terms of quality of life. This paper provides recommendations for improved AWH patient care in regard to mental health.

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression.

The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development.

Use of Hydrogenophaga pseudoflava penetration to quantitatively assess the impact of filtration parameters for 0.2-micrometer-pore-size filters.

Filters rated as having a 0.2-microm pore size (0.2-microm-rated filters) are used in laboratory and manufacturing settings for diverse applications of bacterial and particle removal from process fluids, analytical test articles, and gasses. Using Hydrogenophaga pseudoflava, a diminutive bacterium with an unusual geometry (i.e., it is very thin), we evaluated passage through 0.2-microm-rated filters and the impact of filtration process parameters and bacterial challenge density. We show that consistent H. pseudoflava passage occurs through 0.2-microm-rated filters. This is in contrast to an absence of significant passage of nutritionally challenged bacteria that are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.

In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization.

In situ hybridization is used to survey the tissue-specific and developmental expression of the cloned mouse gene Sparc, coding for a protein homologous to the bovine Ca++-binding protein, osteonectin. High levels of SPARC RNA are found in osteoblasts and odontoblasts. In addition, high grain counts are associated with a variety of other cell types in the embryo and newborn mouse, including parietal endoderm, deciduum, whisker follicles (connective tissue sheath), peripheral nerve trunk, skin (dermis), and stomach (submucosa). Spatially restricted but high levels of SPARC mRNA are also seen in the adult adrenal glands, testis, and ovary. This pattern of differential gene expression demands a reassessment of the function originally proposed for osteonectin, and predicts a much wider role for the protein in a variety of biological processes.

Olfactory-based orientation in artificially imprinted sea turtles.

Sea turtles (Lepidochelys kempi) are being artificially imprinted to Padre Island, Texas, in an effort to establish a new nesting population. These turtles spent more time per exposure in solutions made of Padre Island sand and seawater than in control solutions in a multiple-choice test. This is evidence that sea turtles may detect differences in natural water samples and remember olfactory cues to which they were exposed neonatally and that these differences may affect their orientation behavior. This suggests that imprinting could be used as a conservation technique for establishing new breeding populations of endangered sea turtles.

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins.

BACKGROUND: Coagulation proteins promote neointimal hyperplasia and vascular remodelling after vessel injury, but the precise mechanisms by which they act in vivo remain undetermined. OBJECTIVES: This study, using an injury model in which the neointima is derived from bone marrow (BM)-derived cells, compared inhibition of tissue factor or thrombin on either BM-derived or existing vascular smooth muscle cells. METHODS: Two transgenic (Tg) mouse strains expressing membrane-tethered tissue factor pathway inhibitor (TFPI) or hirudin (Hir) fusion proteins driven by an alpha smooth muscle actin (SMA) promoter were generated (alpha-TFPI-Tg and alpha-Hir-Tg) and the phenotype after wire-induced endovascular injury was compared with that in wild-type (WT) controls. RESULTS: WT mice developed progressive neointimal expansion, whereas injury in either Tg was followed by repair back to a preinjured state. This was also seen when WT mice were reconstituted with BM from Tg mice but not when Tgs were reconstituted with WT BM, in which injury was followed by slowly progressive neointimal expansion. Injection of CD34+ cells from Tg mice into injured WT mice resulted in the accumulation of fusion protein-expressing cells from day 3 onwards and an absence of neointimal hyperplasia in those areas. CONCLUSIONS: Neointimal development after wire-induced endovascular injury in mice was completely inhibited when BM-derived cells infiltrating the damaged artery expressed membrane tethered anticoagulant fusion proteins under an alpha-SMA promoter. These findings enhance our understanding of the pathological role that coagulation proteins play in vascular inflammation.

Safety of nicotine replacement therapy in critically ill smokers: a retrospective cohort study.

Nicotine replacement therapy (NRT) is a common first-line treatment to prevent nicotine withdrawal in smokers. However, available literature reports conflicting results regarding its efficacy and safety in critically ill patients. The objective of this study was to evaluate the relationship between NRT in smokers in the intensive care unit (ICU) and outcomes. This case-control study was conducted in a university-affiliated tertiary hospital ICU. Over a period of five years, 126 active smokers who received transdermal NRT were matched to 126 active smokers who did not receive NRT. The groups were case-matched for sex, age and Acute Physiology and Chronic Health Evaluation II (APACHE II) score. The primary outcome was administration of antipsychotic medication. Secondary outcomes included use of physical restraints, 30-day mortality, and ventilation requirements. Antipsychotic medication was prescribed in 43 (34.1%) patients who received NRT compared to 14 (11.1%) in controls (P <0.01). Physical restraints were used in 37 (29.4%) patients who received NRT, compared to 12 (9.5%) of controls (P <0.01). The 30-day mortality and number of patients intubated was not statistically different between groups. Average length of intubation time was greater in the NRT group (2.56 days; standard deviation 4.16) compared to the control group (1.44 days; standard deviation 2.68) (P=0.012). The use of NRT to prevent nicotine withdrawal in ICU patients is associated with increased use of antipsychotic medication and physical restraint, and with prolonged mechanical ventilation.

Diabetes mellitus in Hopi and Navajo indians. Prevalence of microvascular complications.

In a cross-sectional study of Hopi and Navajo Indians with non-insulin-dependent diabetes mellitus, we found vascular complications to be strongly related to the duration of diabetes. In patients with diabetes of at least 10 yr duration, retinopathy was found in 57%, nephropathy in 40%, peripheral neuropathy in 21%, and peripheral vascular disease in 28%. For the Hopi and Navajo, the duration-specific prevalence rates of microvascular disease were very similar to prevalence rates found in many other populations. Thus we question the concept, based on reports in the late 1960s, that the Hopi and Navajo Indians have hyperglycemia as an isolated chemical abnormality unaccompanied by other manifestations of diabetes mellitus.

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

Factor VII deficiency and the FVII mutation database.

Factor VII (FVII) is a zymogen for a vitamin K-dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 microg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N-terminal domain (Gla) post-translationally modified by gamma-carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C-terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152-Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc. mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild-moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels.

Interferon-gamma modulates prolactin and tissue factor expression in differentiating human endometrial stromal cells.

Cytokines such as interferon-gamma (IFNgamma) released by resident uterine immune cells are thought to influence the expression of differentiated function in the human endometrium. Decidualization of the stromal cell compartment is confined to the superficial endometrial layer in the nonpregnant uterus. To explore the molecular mechanism underlying the spatial expression of the decidual phenotype, the effect of IFNgamma on the induction of two well characterized markers of endometrial stromal (ES) cell differentiation, PRL and tissue factor (TF), has been investigated. IFNgamma antagonizes cAMP-mediated PRL protein and messenger RNA expression in primary ES cell cultures through inhibition of decidual PRL promoter activity. In parallel, IFNgamma stimulates Stat-1 (signal transducer and activator of transcription-1) expression, phosphorylation, and translocation to the nucleus. Exogenously expressed Stat-1 potently represses decidual PRL promoter activation, indicating the potential for the inhibitory effects of IFNgamma to be mediated by Stat-1. We demonstrate that although the coactivator CREB-binding protein/p300 is essential for decidual PRL transcription, this coactivator does not appear to be the target for IFNgamma-mediated repression. By contrast, IFNgamma has little effect on cAMP-mediated TF expression, but induces TF in ES cells not exposed to a decidualizing stimulus. This suggested that in vivo TF expression may not be restricted to decidualizing cells of the superficial layer and was confirmed by immunohistochemical analysis demonstrating intense TF staining in the basal stromal compartment during the regeneration phase of the cycle. The differential sensitivity of decidualization-associated genes to IFNgamma illustrates its potential role as a selective biological response modifier that influences regional function within the endometrium.

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily.

Micronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A-domains) of blood coagulation factor XI (FXI) and plasma pre-kallikrein (PK). We have used mass spectrometry on a recombinant-expressed, putative A-domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A-domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A-domains and are therefore novel members of the PAN module superfamily, which also includes the N-terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A-domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein-protein or protein-carbohydrate interactions during parasite attachment and host cell invasion.

450 million years of hemostasis.

In mammalian blood coagulation, five proteases (factor VII [FVII]; factor IX [FIX]; factor X [FX]; protein C [PC] and prothrombin [PT]) act with five cofactors (tissue factor [TF]; factor V [FV]; factor VIII [FVIII]; thrombomodulin and protein S) to control the generation of fibrin. Biochemical evidence, molecular cloning data and comparative sequence analysis support the existence of all components of this network in all jawed vertebrates, and strongly suggest that it evolved before the divergence of teleosts over 430 million years ago. Phylogenetic analysis of the amino acid sequences of the Gla-EGF1-EGF2-SP domain serine proteases (FVII, FIX, FX, PC) and the A domain-containing cofactors (FV and FVIII) strongly supports the evolution of the blood coagulation network through two rounds of gene duplication, and supports the hypothesis that vertebrate evolution benefited from two global genome duplications. The jawless vertebrates (hagfish and lamprey) that diverged over 450 million years ago have a blood coagulation network involving TF, PT and fibrinogen. Preliminary evidence indicates that they may have a smaller complement of Gla-EGF1-EGF2-SP domain proteins, suggesting the existence of a 'primitive' coagulation system in jawless vertebrates.

Analysis of the consequences of premature termination codons within factor VIII coding sequences.

Inhibitor antibody formation is a complication of factor VIII (FVIII) replacement therapy due to a failure to synthesize sufficient FVIII protein to induce immune tolerance. The incidence of nonsense mutations in inhibitor patients is high, however, this association is variable according to the position of the mutation. We have studied the effect of nonsense mutations on accumulation of FVIII mRNA, protein translation and secretion. Appropriately processed mRNA was detected in cells transfected with wild-type R1966X and R2116X expression constructs and no evidence of nonsense-mediated decay was observed. All constructs directed the translation of detectable intracellular FVIII antigen, however, secreted FVIII was detected only in conditioned media of cells transfected with wild-type cDNA. We have also analyzed ectopic FVIII mRNA transcripts in the lymphocytes of six hemophilia A patients with nonsense mutations (Q139X, R583X, R1941X, R1966X and two unrelated patients with R2116X). FVIII mRNA was detectable in every case. In R1941X and R1966X only normally spliced transcripts were present. In Q139X, R583X and R2116X aberrantly spliced transcripts were observed with two distinct patterns in two individuals with the R2116X mutation. No correlation between mutation, transcript pattern and incidence of inhibitor development was apparent.

Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts.

UNLABELLED: Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. OBJECTIVES: To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. METHODS: Steady-state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse-transcription-polymerase chain reaction. RESULTS: FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP-1 mRNA and protein secretion strongly, IL-8 moderately, and IL-6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR-1 and PAR-3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR-2. CONCLUSIONS: FXa and thrombin induce expression of MCP-1, IL-8 and IL-6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.