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1
Booth, JL, ES Duggan, VI Patel, J. Metcalf, M. Langer, KM Coggeshall, and A. Braun. "ID: 106: ALVEOLAR ESCAPE BY BACILLUS ANTHRACIS SPORES DOES NOT REQUIRE A CARRIER CELL AND IS NOT ALTERED BY LETHAL TOXIN." Journal of Investigative Medicine 64, no. 4 (March 22, 2016): 960.2–961. http://dx.doi.org/10.1136/jim-2016-000120.101.
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RationaleThe lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. B. anthracis spores must escape from the alveolus, pass to the regional lymph nodes, germinate and enter the circulatory system as vegetative bacteria to cause systemic disease. Of the resident lung cells, three have been reported to take up B. anthracis spores: the antigen presenting cells (APC) alveolar macrophages and dendritic cells, and alveolar epithelial cells (AEC). Also, B. anthracis produces the exotoxins lethal factor and protective antigen (PA) which combine to form lethal toxin (LT), a metalloproteinase important in pathogenicity. The roles of carrier cells and the effects of B. anthracis toxins in escape of spores from the alveolus are unclear, especially in humans.MethodsWe employed a human lung organ culture model and a human A549 alveolar epithelial cell culture model, along with fluorescent confocal imaging to quantitate spore partitioning between APC and AEC, and the effects of B. anthracis LT and PA on this process. Cell types were distinguished by positive staining for HLA-DR (APC) and cytokeratin (AEC).ResultsWe found that spores progressed through the lung slice over time, and that spore movement was not dependent on cell internalization. Both free and cell-associated spores moved through slices between 2 and 48 hrs of incubation. However, partitioning of spores between AEC, APC, and the extracellular space did not significantly change over this time. After 2 hrs, 4.7% of spores were in APC; 13.8% in AEC; and 81.5% were not cell-associated. By 48 hrs, 2.9% were in APC; 12.7% were in AEC; and 84.4% were not cell-associated. Spores also internalized in a non-uniform manner, with more variable spore internalization into AEC than into APC. At all incubation times, the majority of cell-associated spores were in AEC, not in APC. PA and LT did not affect transit of the spores through the lung tissue or the distribution of spores into AEC and APC. In A549 cells, spore internalization increased significantly after 24 hrs incubation. However, there was no statistically consistent effects of PA or LT on spore internalization in A549 cells.ConclusionsOverall, our results support a “Jailbreak”-like model of spore escape from the alveolus that involves transient passage of spores, although this occurs through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.
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Kulkarni, Kiran, Ziguo Zhang, Leifu Chang, Jing Yang, Paula C. A. da Fonseca, and David Barford. "Building a pseudo-atomic model of the anaphase-promoting complex." Acta Crystallographica Section D Biological Crystallography 69, no. 11 (October 12, 2013): 2236–43. http://dx.doi.org/10.1107/s0907444913018593.
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The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.
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Zeng, Biqing, Feng Zeng, Heng Yang, Wu Zhou, and Ruyang Xu. "Multi-task learning model for aspect term extraction and aspect polarity classification based on dual-labels." Journal of Intelligent & Fuzzy Systems 39, no. 3 (October 7, 2020): 2763–74. http://dx.doi.org/10.3233/jifs-191047.
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Aspect-based sentiment analysis (ABSA) is a hot and significant task of natural language processing, which is composed of two subtasks, the aspect term extraction (ATE) and aspect polarity classification (APC). Previous researches generally studied two subtasks independently and designed neural network models for ATE and APC respectively. However, it integrates various manual features into the model, which will consume plenty of computing resources and labor. Moreover, the quality of the ATE results will affect the performance of APC. This paper proposes a multi-task learning model based on dual auxiliary labels for ATE and APC. In this paper, general IOB labels, and sentimental IOB labels are equipped to efficiently solve both ATE and APC tasks without manual features adopted. Experiments are conducted on two general ABSA benchmark datasets of SemEval-2014. The experimental results reveal that the proposed model is of great performance and efficient for both ATE and APC tasks compared to the main baseline models.
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Swietlicki, Elzbieta A., Shashi Bala, Jianyun Lu, Anisa Shaker, Gowri Kularatna, Marc S. Levin, and Deborah C. Rubin. "Epimorphin deletion inhibits polyposis in the Apcmin/+ mouse model of colon carcinogenesis via decreased myofibroblast HGF secretion." American Journal of Physiology-Gastrointestinal and Liver Physiology 305, no. 8 (October 15, 2013): G564—G572. http://dx.doi.org/10.1152/ajpgi.00486.2012.
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Interactions between the epithelium and surrounding mesenchyme/stroma play an important role in normal gut morphogenesis, the epithelial response to injury, and epithelial carcinogenesis. The tumor microenvironment, composed of stromal cells including myofibroblasts and immune cells, regulates tumor growth and the cancer stem cell niche. Deletion of epimorphin (Epim), a syntaxin family member expressed in myofibroblasts and macrophages, results in partial protection from colitis and from inflammation-induced colon cancer in mice. We sought to determine whether epimorphin deletion protects from polyposis in the Apc min/+ mouse model of intestinal carcinogenesis. Epim− /− mice were crossed to Apc min/+ mice; Apc min/+ and Apc min/+ /Epim− /− mice were killed at 3 mo of age. Polyp numbers and sizes were quantified in small intestine and colon, and gene expression analyses for pathways relevant to epithelial carcinogenesis were performed. Primary myofibroblast cultures were isolated, and expression and secretion of selected growth factors from Apc min/+ and Apc min/+ /Epim− /− myofibroblasts were examined by ELISA. Small bowel polyposis was significantly inhibited in Apc min/+ /Epim− /− compared with Apc min/+ mice. Apc min/+ /Epim− /− compared with Apc min/+ polyps and adjacent uninvolved intestinal mucosa had increased transforming growth factor-β (TGF-β) expression and signaling with increased P-Smad2/3 expression. Myofibroblasts isolated from Apc min/+ /Epim− /− vs. Apc min/+ mice had markedly decreased hepatocyte growth factor (HGF) expression and secretion. We concluded that Epim deletion inhibits polyposis in Apc min/+ mice, associated with increased mucosal TGF-β signaling and decreased myofibroblast HGF expression and secretion. Our data suggest that Epim deletion reduces tumorigenicity of the stromal microenvironment.
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Luo, Liying, and James S. Hodges. "Constraints in Random Effects Age-Period-Cohort Models." Sociological Methodology 50, no. 1 (February 11, 2020): 276–317. http://dx.doi.org/10.1177/0081175020903348.
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Random effects (RE) models have been widely used to study the contextual effects of structures such as neighborhoods or schools. The RE approach has recently been applied to age-period-cohort (APC) models that are unidentified because the predictors are exactly linearly dependent. However, research has not fully explained how the RE specification identifies these otherwise unidentified APC models. We address this challenge by first making explicit that RE-APC models have greater—not less—rank deficiency than the traditional fixed-effects model, followed by two empirical examples. We then provide intuition and a mathematical proof to explain that for APC models with one RE, treating one effect as an RE is equivalent to constraining the estimates of that effect’s linear component and the random intercept to be zero. For APC models with two REs, the effective constraints implied by the model depend on the true (i.e., in the data-generating mechanism) nonlinear components of the effects that are modeled as REs, so that the estimated linear components of the REs are determined by the true nonlinear components of those effects. In conclusion, RE-APC models impose arbitrary although highly obscure constraints and thus do not differ qualitatively from other constrained APC estimators.
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Arnljots, Björn, David Bergqvist, and Björn Dahlbäck. "Inhibition of Microarterial Thrombosis by Activated Protein C in a Rabbit Model." Thrombosis and Haemostasis 72, no. 03 (1994): 415–20. http://dx.doi.org/10.1055/s-0038-1648881.
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SummaryProtein C is the key component in a natural anticoagulant pathway. After its activation by the thrombin-thrombomodulin complex, it degrades the activated forms of coagulation cofactors VIII and V, which leads to downregulation of the coagulation process. Due to its specific anticoagulant activity, activated protein C (APC) is potentially interesting as an antithrombotic agent. The effect of bovine activated protein C on thrombus formation and haemostasis was investigated in a rabbit model of microarterial thrombosis. Segments of both central ear arteries were prepared and blood-flow interrupted with double vascular clamps.Longitudinal arteriotomies (7 mm) and deep vessel wall trauma (5 mm) were performed, whereafter the arteriotomies were closed with running sutures. Five minutes prior to opening of the clamps (reperfusion), boluses of APC (0.8 mg/kg body weight) or vehicle alone were administered to two groups, each of 10 rabbits, in a blind random fashion. Vessel patency-rates were drastically improved by the administration of APC compared to vehicle. Correspondingly, thrombus weights were significantly lower in the APC group than in the control group. The activated partial thromboplastin time was prolonged to approximately twice the baseline throughout the 2 h observation interval in the APC group. Levels of circulating platelets were unaffected by the APC infusion, but the arteriotomy bleeding times were significantly longer in the APC group. In summary, activated protein C exerted powerful and long-acting antithrombotic effects in a microarterial model of thrombosis in rabbits.
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Zhang, Yongke, and Emma Lees. "Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation." Molecular and Cellular Biology 21, no. 15 (August 1, 2001): 5190–99. http://dx.doi.org/10.1128/mcb.21.15.5190-5199.2001.
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ABSTRACT Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1–153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.
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Hoogendoorn, H., ME Nesheim, and AR Giles. "A qualitative and quantitative analysis of the activation and inactivation of protein C in vivo in a primate model." Blood 75, no. 11 (June 1, 1990): 2164–71. http://dx.doi.org/10.1182/blood.v75.11.2164.2164.
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Abstract A model of Protein C (PC) activation in vivo was used to investigate the complexing of activated PC (APC) with its plasma inhibitors, PC inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT). Chimpanzees were infused with a bolus of activated factor X (F.Xa) together with vesicles of phosphatidylcholine and phosphatidylserine (PCPS). Pre- and post-infusion plasma samples were analyzed using enzyme linked immunosorbent based assays (ELISA) for PC and APC complexes, and immunoblotting of PC from nondenaturing polyacrylamide gel electrophoresis. Within 2 minutes of infusion, a 60% decrease in nonactivated PC zymogen (PCz) levels was observed. This coincided with a precipitous drop in plasma activities of cofactors VIIIa and Va. In contrast, total PC antigen (PCt) levels decreased by only 1%, indicating APC generation. Complexes of APC with both PCI and alpha 1AT were observed on immunoblots, and further identified and quantified using a sandwich ELISA employing antibodies to both PC and these inhibitors. The distribution of APC between these two inhibitors varied with the dose of F.Xa/PCPS infused. At a dose of F.Xa/PCPS of 24.05 pmol and 37.70 nmol/kg, respectively, an initial spike of APC generation, associated with decreases in the levels of factors VIIIa and Va, was noted but dissipated over the next 30 minutes. During this period, APC/inhibitor complexes appeared with the levels of APC-PCI and APC-alpha 1AT reaching 8.5 nmol/L and 2.2 nmol/L by 30 minutes, respectively. In contrast, at a higher dose of F.Xa/PCPS of 36.60 pmol and 56.30 nmol/Kg respectively, complexes of APC-alpha 1AT appeared rapidly and reached a level of 6 nmol/L by 30 minutes postinfusion, whereas APC-PCI complexes were only present at a concentration of 3.4 nmol/L by this time. Additional experiments with lower doses of F.Xa/PCPS suggest that PCI is the preferred inhibitor of APC, but as the availability of this inhibitor becomes limiting, alpha 1AT plays an increasingly crucial role as a secondary inhibitor of endogenously generated APC. Moreover, evidence is presented suggesting the existence of additional inhibitor(s) of APC that may have a role similar to alpha 1AT.
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Hoogendoorn, H., ME Nesheim, and AR Giles. "A qualitative and quantitative analysis of the activation and inactivation of protein C in vivo in a primate model." Blood 75, no. 11 (June 1, 1990): 2164–71. http://dx.doi.org/10.1182/blood.v75.11.2164.bloodjournal75112164.
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A model of Protein C (PC) activation in vivo was used to investigate the complexing of activated PC (APC) with its plasma inhibitors, PC inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT). Chimpanzees were infused with a bolus of activated factor X (F.Xa) together with vesicles of phosphatidylcholine and phosphatidylserine (PCPS). Pre- and post-infusion plasma samples were analyzed using enzyme linked immunosorbent based assays (ELISA) for PC and APC complexes, and immunoblotting of PC from nondenaturing polyacrylamide gel electrophoresis. Within 2 minutes of infusion, a 60% decrease in nonactivated PC zymogen (PCz) levels was observed. This coincided with a precipitous drop in plasma activities of cofactors VIIIa and Va. In contrast, total PC antigen (PCt) levels decreased by only 1%, indicating APC generation. Complexes of APC with both PCI and alpha 1AT were observed on immunoblots, and further identified and quantified using a sandwich ELISA employing antibodies to both PC and these inhibitors. The distribution of APC between these two inhibitors varied with the dose of F.Xa/PCPS infused. At a dose of F.Xa/PCPS of 24.05 pmol and 37.70 nmol/kg, respectively, an initial spike of APC generation, associated with decreases in the levels of factors VIIIa and Va, was noted but dissipated over the next 30 minutes. During this period, APC/inhibitor complexes appeared with the levels of APC-PCI and APC-alpha 1AT reaching 8.5 nmol/L and 2.2 nmol/L by 30 minutes, respectively. In contrast, at a higher dose of F.Xa/PCPS of 36.60 pmol and 56.30 nmol/Kg respectively, complexes of APC-alpha 1AT appeared rapidly and reached a level of 6 nmol/L by 30 minutes postinfusion, whereas APC-PCI complexes were only present at a concentration of 3.4 nmol/L by this time. Additional experiments with lower doses of F.Xa/PCPS suggest that PCI is the preferred inhibitor of APC, but as the availability of this inhibitor becomes limiting, alpha 1AT plays an increasingly crucial role as a secondary inhibitor of endogenously generated APC. Moreover, evidence is presented suggesting the existence of additional inhibitor(s) of APC that may have a role similar to alpha 1AT.
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Fosse, Ethan, and Christopher Winship. "Analyzing Age-Period-Cohort Data: A Review and Critique." Annual Review of Sociology 45, no. 1 (July 30, 2019): 467–92. http://dx.doi.org/10.1146/annurev-soc-073018-022616.
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Age-period-cohort (APC) analysis has a long, controversial history in sociology and related fields. Despite the existence of hundreds, if not thousands, of articles and dozens of books, there is little agreement on how to adequately analyze APC data. This article begins with a brief overview of APC analysis, discussing how one can interpret APC effects in a causal way. Next, we review methods that obtain point identification of APC effects, such as the equality constraints model, Moore-Penrose estimators, and multilevel models. We then outline techniques that entail point identification using measured causes, such as the proxy variables approach and mechanism-based models. Next, we discuss a general framework for APC analysis grounded in partial identification using bounds and sensitivity analyses. We conclude by outlining a general step-by-step procedure for conducting APC analyses, presenting an empirical example examining temporal shifts in verbal ability.
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Von Drygalski, Annette, Andrew J. Gale, Laurent Burnier, Thomas J. Cramer, John H. Griffin, and Laurent O. Mosnier. "An Engineered Factor Fva Prevents Bleeding Induced By Anticoagulant Wild Type Activated Protein C." Blood 122, no. 21 (November 15, 2013): 203. http://dx.doi.org/10.1182/blood.v122.21.203.203.
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Abstract Increased risk of bleeding is observed in patients receiving therapy with a variety of anticoagulant drugs, and there is an unmet need for prohemostatic agents that reduce bleeding risk. In some clinical trials, wild type (wt) APC therapy was associated with an increased risk of serious bleeding. In most animal models of inflammatory injury and disease where APC was beneficial, APC’s cytoprotective effects were responsible for the protective effects of APC therapy whereas its anticoagulant effects were neither required nor contributing. Thus, wt APC’s anticoagulant activities and associated risk of bleeding may be a limiting factor for potential novel wt APC therapies. The availability of a wt APC-anticoagulant specific antidote or reversal agent that does not affect wt APC’s cytoprotective activities would be highly desirable. We hypothesized that superFVa, an engineered FVa-variant that potently normalizes hemostasis in hemophilia, fits the criteria for a prohemostatic biologic that could reduce wt APC-anticoagulant-induced bleeding, and here we test this hypothesis. SuperFVa has enhanced specific activity compared to wt FVa due to an engineered disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (FV(A2-SS-A3)) and, its biological activity is augmented by mutations of the APC cleavage sites (Arg506/306/679Gln). As a result of these modifications, superFVa was found to be resistant to wt APC and normalized hemostatic properties and prevented bleeding in a hemophilic mouse model much more efficiently than wt FVa. In aPTT clotting assays both superFVa and wt FVa dose-dependently normalized clotting times when wt-APC was used to prolong clotting (aPTT > 100 sec). However, superFVa normalized the aPTT at 100-fold lower concentrations compared to wt FVa. In thrombin generation assays using either human or murine plasma, superFVa fully restored thrombin generation at concentrations where wt FVa did not show effects. In an ex vivo whole blood aPTT assay, intravenous (iv) injection of murine recombinant wt APC (0.5 mg/kg) in Balb/c mice doubled clotting times from 30 sec to ∼ 60 sec (n=40). Addition of superFVa (1 nM) to whole blood significantly normalized aPTT clotting times whereas wt FVa failed to show a significant effect. In a tail clip-bleeding model in Balb/c mice, injection (iv) of human recombinant or plasma-derived wt APC induced significant bleeding at 1.25 mg/kg and mean blood loss increased from 3.4 µL/g with saline to 27 µL/g with wt APC treatment. SuperFVa injected (iv 3.5 mg/kg) 2 min prior to wt APC administration reduced bleeding significantly to 9.2 µL/g (n∼10 per group). In another bleeding model, liver laceration was used because it provides important information concerning microvessel-mediated bleeding after acute organ injury. After adaptation and technical modifications of the model used in rats and rabbits for mouse anatomy and validation in hemophilia versus control mice, this model provided a reliable assessment of bleeding with a ∼ 4-fold reduced inter-individual range of blood loss compared to the tail clip-bleeding model. Injection (iv) of human wt APC increased blood loss from 29 µL/g to 49 µL/g, and the excessive bleeding was associated with a ∼40% mortality rate. SuperFVa reduced wt APC-induced bleeding after 20 min significantly to 29 µL/g and abolished bleeding-induced mortality. Remarkably, bleeding patterns in the tail clip and liver laceration models were different when blood loss was determined separately for the 1st and 2nd 10 min after injury. In the tail clip-model wt-APC-induced bleeding during both 1st and 2nd 10 min after tail clip and superFVa decreased blood loss during both phases. In the liver laceration model, most blood loss occurred immediately after injury and bleeding during the 2nd 10 min was less pronounced. In this model, however, superFVa corrected blood loss entirely during the 1st 10 min phase and fully prevented bleeding during the 2nd 10 min phase. Our results provide proof of principle that superFVa is effective in the prevention and reversal of wt APC-induced bleeding. Thus, in addition to improving hemostasis in hemophilia, superFVa protects against bleeding in 2 different mouse models where bleeding was induced by wt APC. Hence, superFVa deserves future consideration for development as a prohemostatic agent in situations where bleeding is a serious risk. Disclosures: No relevant conflicts of interest to declare.
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Kerschen, Edward J., Brian C. Cooley, Francis J. Castellino, John H. Griffin, and Hartmut Weiler. "Protective Effect of Activated Protein C in Murine Endotoxemia: Mechanism of Action." Blood 106, no. 11 (November 16, 2005): 26. http://dx.doi.org/10.1182/blood.v106.11.26.26.
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Abstract Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expression of the endothelial protein C receptor (EPCR). Recombinant mutant murine APC with greatly reduced anticoagulant potency was as effective as wild type murine APC in reducing mortality of mice subjected to LPS-induced septicemia. Mice homozygous for the Leiden polymorphism in the factor V (fV) gene, which renders coagulation factor V partially resistant to the anticoagulant effect of APC secondary to blocked fV proteolysis at R504 (R506 in humans), were refractory to the therapeutic benefit conveyed by administration of recombinant wild type mouse APC. In summary, these findings provide evidence that the therapeutic efficacy of recombinant APC is predominantly based on the ability of APC to interact with the endothelial protein C receptor, and that the anticoagulant activity of APC is not sufficient for achieving protection against mortality in a mouse model of endotoxemia. On the other hand, cleavage of fV at R506 appears necessary for retaining therapeutic efficacy in carriers of the fV Leiden allele.
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Gruber, A., JH Griffin, LA Harker, and SR Hanson. "Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model." Blood 73, no. 3 (February 15, 1989): 639–42. http://dx.doi.org/10.1182/blood.v73.3.639.639.
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Abstract The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.
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Gruber, A., JH Griffin, LA Harker, and SR Hanson. "Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model." Blood 73, no. 3 (February 15, 1989): 639–42. http://dx.doi.org/10.1182/blood.v73.3.639.bloodjournal733639.
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The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.
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Linden, M. D., M. Schneider, and W. N. Erber. "Factor VLEIDEN and cardiopulmonary bypass: investigation of haemostatic parameters and the effect of aprotinin using an ex vivo model." Perfusion 16, no. 6 (December 2001): 476–84. http://dx.doi.org/10.1177/026765910101600607.
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It has been suggested that aprotinin results in significantly increased risk for perioperative thrombotic complications in patients with Factor VLEIDEN (F5L) due to its ability to competitively inhibit activated protein C (APC) function in vitro. No clinical studies have been performed to assess the effect of aprotinin on APC function of F5L in vivo. We developed an ex vivo model to mimic the effects of cardiopulmonary bypass with the exclusion of the patient in order to assess APC function. Blood from normal ( n = 2) and F5L heterozygous donors ( n = 2) was treated with aprotinin or placebo (saline). The blood was heparinized, added to the prime and circulated at 2 l/min through a modified cardiopulmonary bypass circuit. After 60 min of circulation, the heparin was neutralized with protamine sulfate. Blood samples, drawn at specific time points, were analysed for APC ratio. Results showed a decrease in APC ratio for both F5L and normal bloods with the addition of aprotinin (18% and 40%, respectively). APC ratios also decreased with the commencement of extracorporeal circulation for all bloods, resulting in an APC ratio of 1.35 in normal placebo-treated blood and 0.67 in F5L placebo-treated blood. The combined effect of aprotinin and extracorporeal circulation resulted in APC ratios of 0.90 for normal blood and 0.63 for F5L blood, corresponding to a severe dysfunction of APC intraoperatively (reference range 1.9-4.0). The data from this model predict an increased risk of perioperative thrombosis due to inhibition of APC function in cardiac surgical patients heterozygous for the F5L mutation. Aprotinin further compounds the severity of APC dysfunction, though the effect is more severe in normal blood. The ex vivo model employed was an effective tool for the investigation of the haemostatic effect of aprotinin. This model may be exploited for other applications such as the investigation of novel or emerging haemostatic agents prior to clinical trial.
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Mosnier, Laurent O., Jose A. Fernandez, Antonella Zampolli, Xia V. Yang, Zaverio M. Ruggeri, and John H. Griffin. "In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants." Blood 110, no. 11 (November 16, 2007): 2704. http://dx.doi.org/10.1182/blood.v110.11.2704.2704.
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Abstract Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC’s anticoagulant activity vs. APC’s direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH2006, J Exper Med, 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC’s anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.
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Souris, Jeffrey S., Hannah J. Zhang, Urszula Dougherty, Nai-Tzu Chen, Joseph V. Waller, Leu-Wei Lo, John Hart, Chin-Tu Chen, and Marc Bissonnette. "A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase." Carcinogenesis 40, no. 11 (March 12, 2019): 1376–86. http://dx.doi.org/10.1093/carcin/bgz050.
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AbstractAlthough valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene — either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model’s small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors — unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.
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Kerschen, Edward J., Brian C. Cooley, Francis J. Castellino, Shaun R. Coughlin, Jose A. Fernandez, John H. Griffin, and Hartmut Weiler. "Mechanisms for Mortality Reduction by Activated Protein C in Severe Sepsis." Blood 108, no. 11 (November 16, 2006): 1. http://dx.doi.org/10.1182/blood.v108.11.1.1.
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Abstract Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC’s beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over < 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50% to 0–10% at LD-50 LPS doses). APC treatment did not alter the extent of circulating inflammatory cytokine levels at 3 or 24 hr after endotoxin exposure. The survival benefit conferred by wt APC infusions was abolished in mice with genetically reduced levels of endothelial protein C receptor (EPCR) (< 10% of normal) or in mice genetically lacking protease-activated receptor-1 (PAR-1). Murine APC variants with either 3 or 5 Ala substitutions, 3K3A-APC (KKK192-194AAA) or 5A-APC (RR230/231AA + KKK192-194AAA) that had reduced anticoagulant activity (25 % and < 10 % of wt APC, respectively), but normal cytoprotective activities, were as effective as wt APC in reducing mortality after LPS challenge. A murine APC variant lacking proteolytic activity (active site S360A) did not enhance survival after LPS, showing a requirement for APC’s enzymatic activity. Thus, the survival-promoting efficacy of APC in this model requires the enzymatic active site of APC and the presence of two receptors, EPCR and PAR-1, that are known to mediate APC’s in vitro beneficial cytoprotective effects on cells. In a whole bacteria sepsis model, when APC was given to mice at the time of initiation of peritoneal Staphylococcus aureus-induced sepsis and again at 24 hr, wt APC surprisingly increased mortality (100% mortality vs. 50% at LD-50 bacteria dose). In contrast, when 3K3A-APC or 5A-APC variants with attenuated anticoagulant activity was given at 0 and 24 hr, they prevented mortality due to bacterial sepsis (0–10% vs. 50% mortality for saline control at LD-50 dose). This implies that APC’s anticoagulant action might impair beneficial coagulation-dependent host defense mechanisms in early stages of bacterial sepsis whereas the 5A-APC variant, with very low anticoagulant activity but normal cytoprotective activity, might provide beneficial cellular effects to help prevent death during bacterial sepsis. In summary, the full anticoagulant activity of APC is not required for protection against mortality in each of these models. These results highlight the importance of the cellular protein C pathway for APC therapy and suggest that APC variants with reduced anticoagulant action but normal potency for beneficial direct cellular effects merit further evaluation for sepsis therapy.
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Uneno, Yu, Tadayuki Kou, Masashi Kanai, Michio Yamamoto, Peng Xue, Yukiko Mori, Yasushi Kudo, et al. "Prognostic model for survival in patients with advanced pancreatic cancer receiving palliative chemotherapy." Journal of Clinical Oncology 33, no. 3_suppl (January 20, 2015): 248. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.248.
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248 Background: The prognosis of patients with advanced pancreatic cancer (APC) is extremely poor. Several clinical and laboratory factors have been known to be associated with prognosis of APC patients. However, there are few clinically available prognostic models predicting survival in APC patients receiving palliative chemotherapy. Methods: To construct a prognostic model to predict survival in APC patients receiving palliative chemotherapy, we analyzed the clinical data from 306 consecutive patients with pathologically confirmed APC who received palliative chemotherapy. We selected six independent prognostic factors which remained independent prognostic factors after multivariate analysis. Thereafter, we rounded the regression coefficient (β) for each independent prognostic factor derived from the Cox regression equation (HR = eβ) and developed a prognostic index (PI). Results: Developed prognostic index (PI) was as follows: PI = 2 (if performance status score 2–3) + 1 (if metastatic disease) + 1 (if initially unresectable disease) + 1 (if carcinoembryonic antigen level ≥5.0 ng/ml) + 1 (if carbohydrate antigen 19-9 level ≥1000 U/ml) + 2 (if neutrophil–lymphocyte ratio ≥5). The patients were classified into three prognostic groups: favorable (PI 0–1, n = 73), intermediate (PI 2–3, n = 145), and poor prognosis (PI 4–8, n = 88). The median overall survival for each prognostic group was 16.5, 12.3 and 6.2 months, respectively, and the 1-year survival rates were 67.3%, 51.3%, and 19.1%, respectively (P < 0.01). The c index of the model was 0.658. This model was well calibrated to predict 1-year survival, in which overestimation (2.4% and 0.2% in the favorable and poor prognosis groups, respectively) and underestimation (3.6% in the intermediate prognosis group) were observed. Conclusions: This prognostic model based on readily available clinical factors would help clinicians in estimating the overall survival in APC patients receiving palliative chemotherapy.
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Lazic, Divna, Abhay P. Sagare, Angeliki M. Nikolakopoulou, John H. Griffin, Robert Vassar, and Berislav V. Zlokovic. "3K3A-activated protein C blocks amyloidogenic BACE1 pathway and improves functional outcome in mice." Journal of Experimental Medicine 216, no. 2 (January 15, 2019): 279–93. http://dx.doi.org/10.1084/jem.20181035.
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3K3A-activated protein C (APC), a cell-signaling analogue of endogenous blood serine protease APC, exerts vasculoprotective, neuroprotective, and anti-inflammatory activities in rodent models of stroke, brain injury, and neurodegenerative disorders. 3K3A-APC is currently in development as a neuroprotectant in patients with ischemic stroke. Here, we report that 3K3A-APC inhibits BACE1 amyloidogenic pathway in a mouse model of Alzheimer’s disease (AD). We show that a 4-mo daily treatment of 3-mo-old 5XFAD mice with murine recombinant 3K3A-APC (100 µg/kg/d i.p.) prevents development of parenchymal and cerebrovascular amyloid-β (Aβ) deposits by 40–50%, which is mediated through NFκB–dependent transcriptional inhibition of BACE1, resulting in blockade of Aβ generation in neurons overexpressing human Aβ-precursor protein. Consistent with reduced Aβ deposition, 3K3A-APC normalized hippocampus-dependent behavioral deficits and cerebral blood flow responses, improved cerebrovascular integrity, and diminished neuroinflammatory responses. Our data suggest that 3K3A-APC holds potential as an effective anti-Aβ prevention therapy for early-stage AD.
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Livnat, Tami, Yael Nisgav, Ivan Budnik, Yehonatan Weinberger, Rima Dardik, Gili Kenet, and Dov Weinberger. "Activated Protein C Induces Stabilization of the Outer Retinal Barrier and Inhibits Laser Induced Choroidal Neovascularization in a Murine Model." Blood 132, Supplement 1 (November 29, 2018): 73. http://dx.doi.org/10.1182/blood-2018-99-111375.
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Abstract Introduction: Age-related macular degeneration (AMD) is considered one of the main causes of severe vision loss in older adults. Wet AMD is characterized by choroidal neovascular membrane developed under or into the sensory retina, breaking the outer blood-retina barrier (oBRB) and causing leakage and bleeding. Protein C (PC) is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts diverse homeostatic cell signaling pathways apart from its anticoagulant activity. APC activities are mediated mainly through endothelial PC receptor (EPCR) and protease activated receptor (PAR)-1. APC was recently shown to act as a novel ligand for Tie2, a tyrosine kinase receptor playing an important role in angiogenesis. Thus, APC may modulate pathological angiogenesis in relevant ocular conditions, such as choroidal neovascularization (CNV), via its interaction with Tie2. To the best of our knowledge, there are no studies addressing the potential use of APC as oBRB stabilizer and CNV inhibitor. Aim: To assess the protective effect of APC as a retinal barrier stabilizer and its ability to reduce CNV growth, leakage and penetration from the choroid into the sensory retina. Moreover, to study the role of Tie2 receptor upon APC mode of action in the retina. Methods: CNV development was induced in C57BL/6J mice by laser applications. Immediately following CNV induction APC (0.1-10 µg/µl) was injected intravitrealy. Seven to 14 days later, the retina was separated from the underlying tissues and the 2 segments were flattened: sclera-choroid-retinal pigment epithelium (RPE) segment and the photoreceptors-sensory retina segment. Blood vessels were stained using CD31 (an endothelial marker) or FITC-dextran and analyzed by using a confocal microscopy. Fluorescein angiography (FA) was performed to detect leakage from CNV. The ability of APC to stabilize the oBRB and prevent leakage was studied using ARPE-19 tissue culture (simulating the oBRB). FITC-Dextran leakage through the cultured cell monolayers was measured and the tight junction protein, Zonula occludens-1 (ZO1) cellular distribution was followed using immunostaining. Tie2 Kinase inhibitor and anti-Tie2 antibodies were used for Tie2 inhibition in the murine and cell culture models, respectively. Results: Quantitative analysis of the CNV area and volume revealed that 7-14 days after laser applications, APC treatment resulted in a statistically significant reduction in CNV growth, area and volume as compared to saline. Moreover, APC inhibited the penetration of CNV from the choroid throughout the RPE into the sensory retina in a dose-dependent manner. Figure 1 summarize the quantitative assessment of APC suppression of blood vessel penetration from the choroid (Fig.1A) to the sensory retina segment (Fig.1B). FA analysis showed that APC significantly suppressed the leakage of fluorescein from the laser lesion and preserved the retinal architecture. Incubation of RPE cells with APC resulted in dose dependent translocation of ZO1 to the cell membrane region, accompanied by reduction in permeability throughout the RPE monolayer. ZO1 translocation formed a typical ZO1 honeycomb pattern that is characteristic for the stabilization of tight junctions (Fig.1C). Inhibition of Tie2, in the laser induced CNV mice model and in RPE cell culture, significantly decreased APC protective activities in both models. Conclusions: Several Chorio-retinal pathologies are accompanied by damage to the oBRB and pathological growth of CNV. Our present data provide evidence that APC can protect the choroid and the retina from CNV growth, can suppress leakage from the newly formed choroidal blood vessels and may act as oBRB stabilizer. The protective function of APC in the eye provides direction for new strategies to treat chorio-retinal diseases affected by abnormal oBRB function and CNV growth. Our preliminary results warrant further investigation to extend the data regarding the potential use of APC as a novel anti-CNV treatment and oBRB protector. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Wang, Chongkai, Ching Ouyang, Jaideep Singh Sandhu, Michael Kahn, and Marwan Fakih. "Wild-type APC and prognosis in metastatic colorectal cancer." Journal of Clinical Oncology 38, no. 4_suppl (February 1, 2020): 223. http://dx.doi.org/10.1200/jco.2020.38.4_suppl.223.
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223 Background: Somatic mutations at adenomatous polyposis coli ( APC) gene, found in ~75% of colorectal cancers (CRC), are under-represented in microsatellite instable (MSI-H) tumors. While several studies have suggested worse outcomes for CRC patients (pts) with wild-type APC ( APC-WT), the prognostic implication of this genomic alteration in metastatic CRC (mCRC) is not well defined. Methods: APC prognostic value was evaluated in 331 stage IV microsatellite stable (MSS) CRC pts treated in our institution. Next-generation genomic analysis (FoundationOne) was used to characterize the molecular characteristics of APC-WT and mutant APC ( APC-MT) pts. Findings were validated on a public database of stage IV colon cancer from MSKCC. Results: APC-WT was present in 26% of mCRC patients. In comparison to APC-MT population (n = 244), APC-WT pts (n = 87) tended to be younger (median age: 49 vs. 58 years), right-sided (44% vs. 24%), BRAF-V600E mutated (25% vs. 5%), p53 WT (38% vs. 21%) and RAS WT (66% vs. 53%). APC-WT tumors were associated with other Wnt activating alterations ( CTNNB1, FBXW7, RNF43, ARID1A and SOX9). Among those, RNF43 and CTNNB1 were more significantly represented in the APC-WT vs APC-MT population (12% vs 1% and 11% vs 3%, respectively). APC-WT pts had a worse overall survival (OS) than APC-MT pts (30 vs 48 months, HR = 1.809, 95% CI 1.260-2.596, p < 0.0001). Using a multivariate model correcting for primary tumor location, RAS and BRAF status, APC-WT was predictive of poor survival (HR = 1.7, p = 0.001) in our data set. The prognostic implication of APC-WT on OS were confirmed further in a similar multivariate model of 433 stage IV pts from MSKCC public database (HR = 1.6, P = 0.01). Conclusions: APC-WT is associated with poor OS in MSS mCRC regardless of RAS, BRAF status. Compared with APC-MT mCRC tumors, APC-WT tumors were associated with other activating alterations of Wnt pathway, including RNF43 and CTNBB1.
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Teixeira da Silva, Jaime A. "Three new suggested guidelines for increased transparency regarding open access article processing charges (APCs)." Epistēmēs Metron Logos, no. 4 (July 21, 2020): 4. http://dx.doi.org/10.12681/eml.24208.
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The article processing charge (APC) lies at the heart of the gold open access (GOA) business model. Small and larger society-based, as well as commercial publishers, rely – to different extents – on the APC and the GOA model to thrive. There is wide debate regarding what amount of APC is considered to be exploitative, and the issue of low APCs is often erroneously associated with “predatory” OA publishing. Independent of this debate, there is still, surprisingly, considerable opacity related to the APC used to cover the cost of GOA. In a bid to increase transparency, a simple 3-point plan at increasing academic and financial transparency of authors and journals/publishers regarding APCs is proposed: 1) indicate which author paid the APC in multi-author papers; 2) indicate the value of the APC paid; 3) provide online proof or certification of APC payment, including the indication of any discounts or waivers.
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Tucker, Erik I., Melani Helm, Owen JT McCarty, Sawan Hurst, David Gailani, and Andras Gruber. "Factor XI Inhibitor Antibody Treatment Improves Survival In a Murine Polymicrobial Sepsis Model." Blood 116, no. 21 (November 19, 2010): 820. http://dx.doi.org/10.1182/blood.v116.21.820.820.
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Abstract Abstract 820 Sepsis results in a systemic inflammatory state that is frequently accompanied by intravascular coagulation and fibrinolysis, resulting in a coagulopathy with thrombotic and hemorrhagic components (disseminated intravascular coagulation– DIC). We have shown that the plasma coagulation protease factor XI (FXI) contributes substantially to experimental thrombus formation in baboons and mice, but does not appear to be essential for hemostasis. These results are supported by studies in human populations that show FXI deficiency confers a decreased risk of thrombotic ischemic stroke and deep venous thrombosis, while the associated bleeding diathesis is often mild. FXI appears to contribute to lethal consumptive coagulopathy in protein C deficient mice, while FXI deficiency reduces DIC and prolongs survival of surgical cecal-ligation and puncture (CLP)-induced abdominal sepsis in mice. Taken together, these data suggest that FXI may be an important contributor to the response leading to DIC in sepsis, and that inhibiting FXI may be a safer therapeutic alternative in this setting to activated protein C (APC), which can exacerbate hemorrhage. To investigate the contribution of FXI to consumptive coagulopathy and mortality in sepsis we used the standard CLP polymicrobial sepsis model. To inhibit FXI in the mouse, we developed a new murine anti-mouse FXI monoclonal antibody (14E11) that targets the Apple 2 domain of FXI, and has been shown in vitro to inhibit the activation of FXI by factor XIIa, while not significantly inhibiting activation by thrombin. A single injection of 14E11 (4 mg/kg, SC) prolonged the aPTT of mice up to 3-fold for 48 hrs. Following CLP, the abdomen was closed and mice were treated with vehicle (PBS, SC), APC (6 mg/kg, SC), or 14E11 (4 mg/kg, SC) (n=20 for each group). Overall survival was 45% for vehicle, 15% for APC, and 80% for 14E11 treated mice (P<0.001 for 14E11 vs. both APC and vehicle). 24 hrs after CLP, platelet count in vehicle, APC, and 14E11 treated mice (n=8 each) were lower by 24±7%, 25±5%, and 12±6%, and leukocyte counts were lower by 51±15%, 42±14%, and 43±13% respectively, compared with baseline. Thrombin/antithrombin complex levels were higher in the vehicle treated group 24 hrs after CLP (5.0±1.2 μg/L) compared with 2.2±0.2 μg/L in normal healthy mice (P<0.05), while APC and 14E11 treated groups showed only moderately elevated TAT levels (2.6±0.2 and 2.7±0.4 μg/L, respectively). The pharmacological effects of 14E11 later in the course of sepsis and the apparent poor outcome of early APC treatment remain to be evaluated. In a separate cohort (n=12 each), tail-clip bleeding times were 12.8±1.0, 17.9±1.8, and 12.1±1.7 min for vehicle, APC, and 14E11 treated animals respectively (P<0.05 for APC vs. vehicle and 14E11) 30min after injection. In summary, the outcome was better for 14E11 treated mice in CLP-induced sepsis compared to vehicle or APC treatment, and the data indicate that consumptive coagulopathy may be less severe following FXI inhibition. Furthermore, mice treated with 14E11 showed no increase in bleeding compared with vehicle treatment, while APC significantly prolonged the tail bleeding time. The results suggest that therapeutic inhibition of FXI, by specifically inhibiting FXI activation by FXIIa, could be beneficial in treating sepsis-related DIC. It is also possible that inhibition of FXI may limit DIC with a lower risk of exacerbating bleeding when compared to anticoagulant therapies such as heparin or APC. Disclosures: Tucker: Aronora,LLC: Employment, Equity Ownership, Patents & Royalties. Helm:Aronora,LLC: Employment. Gruber:Aronora,LLC: Consultancy, Equity Ownership, Patents & Royalties.
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Kwak, Kyeongmin, Sung-il Cho, and Domyung Paek. "Future Incidence of Malignant Mesothelioma in South Korea: Updated Projection to 2038." International Journal of Environmental Research and Public Health 18, no. 12 (June 19, 2021): 6614. http://dx.doi.org/10.3390/ijerph18126614.
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Malignant mesothelioma (MM) is a cancer that is largely caused by exposure to asbestos. Although asbestos is no longer used in South Korea, the incidence of MM continues to increase due to its long latent period. We aimed to update the previous prediction of MM incidence until 2038. We predicted the incidence of MM over the next 20 years (2019–2038) in South Korea using Møller’s age–period–cohort (APC) model and a Poisson regression model based on asbestos consumption. The APC model predicted that the crude incidence rate would increase sharply in men and slowly in women. Despite the sex discrepancy in the rate of increase, the incidence rate for both sexes is expected to continue increasing until 2038. In the Poisson model, the crude incidence rate was predicted to increase continuously until 2038, and far more cases of MM were predicted to occur compared with the results of the APC model. When compared with actual incidence data, the APC model was deemed more suitable than the Poisson model. The APC model predicted a continuous increase over the next 20 years with no peak, suggesting that the incidence of MM will continue to rise far into the future.
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Vleminckx, Kris, Ellen Wong, Kathy Guger, Bonnee Rubinfeld, Paul Polakis, and Barry M. Gumbiner. "Adenomatous Polyposis Coli Tumor Suppressor Protein Has Signaling Activity in Xenopus laevis Embryos Resulting in the Induction of an Ectopic Dorsoanterior Axis." Journal of Cell Biology 136, no. 2 (January 27, 1997): 411–20. http://dx.doi.org/10.1083/jcb.136.2.411.
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Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein β-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic β-catenin expression. In fact, axis induction by APC required the availability of cytosolic β-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of β-catenin, indicating that it has direct positive signaling activity in addition to its role in β-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/β-catenin signaling pathway, either upstream of, or in conjunction with, β-catenin.
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Lane, Steven W., Stephen M. Sykes, Fatima Al-Shahrour, Sebastian Shterental, Mahnaz Paktinat, Cristina Lo Celso, Jonathan L. Jesneck, Benjamin L. Ebert, David A. Williams, and D. Gary Gilliland. "The Apcmin mouse has altered hematopoietic stem cell function and provides a model for MPD/MDS." Blood 115, no. 17 (April 29, 2010): 3489–97. http://dx.doi.org/10.1182/blood-2009-11-251728.
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Abstract Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apcmin mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).
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Livnat, Tami, Yehonatan Weinberger, José A. Fernández, Alaa Bashir, Gil Ben-David, Dahlia Palevski, Sarina Levy-Mendelovich, et al. "Activated Protein C (APC) and 3K3A-APC-Induced Regression of Choroidal Neovascularization (CNV) Is Accompanied by Vascular Endothelial Growth Factor (VEGF) Reduction." Biomolecules 11, no. 3 (February 26, 2021): 358. http://dx.doi.org/10.3390/biom11030358.
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The activated protein C (APC) ability to inhibit choroidal neovascularization (CNV) growth and leakage was recently shown in a murine model. A modified APC, 3K3A-APC, was designed to reduce anticoagulant activity while maintaining full cytoprotective properties, thus diminishing bleeding risk. We aimed to study the ability of 3K3A-APC to induce regression of CNV and evaluate vascular endothelial growth factor (VEGF) role in APC’s activities in the retina. CNV was induced by laser photocoagulation on C57BL/6J mice. APC and 3K3A-APC were injected intravitreally after verification of CNV presence. CNV volume and vascular penetration were evaluated on retinal pigmented epithelium (RPE)-choroid flatmount by fluorescein isothiocyanate (FITC)-dextran imaging. VEGF levels were measured using immunofluorescence anti-VEGF staining. We found that 3K3A-APC induced regression of pre-existing CNV. VEGF levels, measured in the CNV lesion sites, significantly decreased upon APC and 3K3A-APC treatment. Reduction in VEGF was sustained 14 days post a single APC injection. As 3K3A-APC retained APCs’ activities, we conclude that the anticoagulant properties of APC are not mandatory for APC activities in the retina and that VEGF reduction may contribute to the protective effects of APC and 3K3A-APC. Our results highlight the potential use of 3K3A-APC as a novel treatment for CNV and other ocular pathologies.
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Kowalska, M. Anna, Shawn A. Mahmud, Michele P. Lambert, Mortimer Poncz, and Arne Slungaard. "Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge." Blood 110, no. 6 (September 15, 2007): 1903–5. http://dx.doi.org/10.1182/blood-2007-03-081901.
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AbstractPharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4−/−) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C–deficient (PC+/−) mice. Transgenic mice overexpressing human PF4 (hPF4+) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC+/− mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4+ and PC+/−/hPF4+ mice had improved survival. Further, infusion of hPF4+ platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting.
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van ’t Veer, Cornelis, Joris Roelofs, Bruce Gerlitz, Brian Grinnell, Marcel Levi, Tom der Poll, and Marcel Schouten. "Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia." Thrombosis and Haemostasis 106, no. 12 (2011): 1189–96. http://dx.doi.org/10.1160/th11-06-0438.
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SummaryRecombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae. Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of throm-bin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.
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Nold-Petry, Claudia, Doris Fischer, Bernd Richter, Roman Blaheta, Josef Pfeilschifter, Heiko Muhl, Dietmar Schranz, Alex Veldman, and Marcel Nold. "Activated protein C downregulates p38 mitogen-activated protein kinase and improves clinical parameters in an in-vivo model of septic shock." Thrombosis and Haemostasis 98, no. 11 (2007): 1118–26. http://dx.doi.org/10.1160/th07-01-0052.
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SummaryDespite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasmaTNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC’s clinical success may, in part, be due to p38MAPK inhibition.
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Borges, Anne Karin da Mota, Adalberto Miranda-Filho, Sérgio Koifman, and Rosalina Jorge Koifman. "Thyroid Cancer Incidences From Selected South America Population-Based Cancer Registries: An Age-Period-Cohort Study." Journal of Global Oncology, no. 4 (December 2018): 1–11. http://dx.doi.org/10.1200/jgo.17.00024.
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Purpose The incidence of thyroid cancer (TC) has increased substantially worldwide. However, there is a lack of knowledge about age-period-cohort (APC) effects on incidence rates in South American countries. This study describes the TC incidence trends and analyzes APC effects in Cali, Colombia; Costa Rica; Goiânia, Brazil; and Quito, Ecuador. Materials and Methods Data were obtained from the Cancer Incidence in Five Continents series, and the crude and age-standardized incidence rates were calculated. Trends were assessed using the estimated annual percentage change, and APC models were estimated using Poisson regression for individuals between age 20 and 79 years. Results An increasing trend in age-standardized incidence rates was observed among women from Goiânia (9.2%), Costa Rica (5.7%), Quito (4.0%), and Cali (3.4%), and in men from Goiânia (10.0%) and Costa Rica (3.4%). The APC modeling showed that there was a period effect in all regions and for both sexes. Increasing rate ratios were observed among women over the periods. The best fit model was the APC model in women from all regions and in men from Quito, whereas the age-cohort model showed a better fit in men from Cali and Costa Rica, and the age-drift model showed a better fit among men from Goiânia. Conclusion These findings suggest that overdiagnosis is a possible explanation for the observed increasing pattern of TC incidence. However, some environmental exposures may also have contributed to the observed increase.
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Tanner, Scott M., Joseph G. Daft, Stephanie A. Hill, Colin A. Martin, and Robin G. Lorenz. "Altered T-Cell Balance in Lymphoid Organs of a Mouse Model of Colorectal Cancer." Journal of Histochemistry & Cytochemistry 64, no. 12 (October 23, 2016): 753–67. http://dx.doi.org/10.1369/0022155416672418.
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The adenomatous polyposis coli ( APC) gene is a known tumor suppressor gene, and mice with mutations in Apc (ApcMin/+) spontaneously form multiple intestinal neoplasms. In this model of human colorectal cancer (CRC), it has been reported that CD4+ T-cell-derived interleukin 17 (IL-17) promotes intestinal tumor development, but it is not known if the Apc mutation actually directly alters T-cell function and subsequently tumor immunosurveillance. To investigate the ApcMin/+ mutation on T-cell function, flow cytometric, histochemical, and immunofluorescent studies on both wild-type (Apc+/+) and ApcMin/+ mice were performed. We identified decreased levels of interferon gamma (IFN-γ+)IL-17+ double-positive CD4+ cells in the mesenteric lymph nodes and Peyer’s patches of ApcMin/+ mice. In addition, altered levels of CD8+ cells, and changes in CD8+ production of IFN-γ and granzyme B were observed. These T-cell alterations did modify tumor immunosurveillance, as the adoptive transfer of splenocytes from ApcMin/+ animals into a chemically induced CRC model resulted in the inability to prevent epithelial dysplasia. These results suggest an altered T-cell balance in ApcMin/+ mice may disrupt intestinal homeostasis, consequently limiting intestinal tumor immunosurveillance.
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Pellequer, Jean-Luc, Andrew Gale, Elizabeth Getzoff, and John Griffin. "Three-dimensional Model of Coagulation Factor Va Bound to Activated Protein C." Thrombosis and Haemostasis 84, no. 11 (2000): 849–57. http://dx.doi.org/10.1055/s-0037-1614127.
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SummaryA complete molecular model of blood coagulation factor Va (FVa) bound to anticoagulant activated protein C (APC) and to a phospholipid membrane was constructed. The three homologous A domains and the two homologous C domains of FVA were modeled based on the X-ray crystallographic structures of ceruloplasmin and C2 domain of factor V, respectively. The final arrangement of the five domains in the complete FVa model bound to a membrane incorporated extensive published experimental data. FVa binds the phospholipid membrane through its C2 domain while the A-domain trimer is located from 40 through 100 Å above the membrane plane. From our model we infer a probable role for metal ions at the interface between FVa light and heavy chains, provide an explanation for the slower APC cleavage at Arg306 relative to Arg506, and predict specific interactions between positively and negatively charged exosites in APC and FVa, respectively.
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Meledeo, Michael A., James E. Campbell, Armando C. Rodriguez, Melanie V. Valenciana, and Andrew P. Cap. "Activated Protein C Levels Found In Trauma Patients Are Insufficient To Inactivate Platelet Factor Va and Produce Coagulopathy In An In Vitro Model." Blood 122, no. 21 (November 15, 2013): 4767. http://dx.doi.org/10.1182/blood.v122.21.4767.4767.
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Background A subset of severe trauma victims suffering from uncontrolled hemorrhage present to the emergency room with an acute coagulopathy of trauma (ACoT). This condition occurs within the first 30 min following injury, is not the result of resuscitation-associated dilutional effects, and is defined by an INR > 1.2. The mechanisms behind this coagulopathy have not been completely identified, but recent studies posit that the exuberant activation of protein C is a key contributor to ACoT. Protein C is activated through the interaction of thrombin and thrombomodulin on the endothelial surface. Activated protein C (APC) has anti-inflammatory and cytoprotective functions, in addition to regulating coagulation by inactivating factor Va (FVa). Evidence that APC is implicated in ACoT includes: 1) circulating APC levels are elevated 3-5 fold in patients with high injury severity scores (>15) and base deficit (>6); 2) high APC levels (>6 ng/ml, or 105 pM) in these patients are correlated with an increase in prothrombin time (PT) and partial thromboplastin time [Cohen et al. Ann Surg 2012; 255(2): 379-85]. FVa exists in a soluble plasma fraction and in a platelet-associated fraction (in the membrane or alpha-granules). The platelet pool of FVa is more resistant to APC degradation [Camire et al. J Biol Chem 1995; 270(35): 20794-800] and is sufficient to maintain thrombin generation even in patients with severe congenital FV-deficiencies [Duckers et al. Blood 2010; 115(4): 879-86]. We hypothesize that healthy platelets in sufficient quantity will serve as an effective promoter of hemostasis in patients with ACoT, overcoming the effects of APC. Methods and Results We studied the effects of APC on coagulation in commercially obtained FV-deficient plasma (FVdp) and platelets/plasma from healthy volunteers. Tests included prothrombin time, turbidimetric assessment of fibrin cross-linking, and thromboelastography (TEG). Prothrombin time in platelet-free plasma or FVdp subjected to exogenous APC display a dose-dependent response where clotting is only significantly delayed at APC >1 nM, much higher than the systemic concentrations found in the ACoT patients (105pM) or even in subjects treated with drotrecogin alfa, a recombinant APC product intended for the treatment of sepsis (steady-state concentration was found to be ∼790pM) [Macias et al. Clin Pharmacol Ther 2002; 72(4): 391-402]. Examining fibrin crosslinking in a static 96-well turbidimetric plate assay demonstrated that concentrations of APC of 1nM or below had no effect on the initiation time, rate, or strength of clotting in FVdp or PFP from healthy volunteers. Titration of plasma FV into FVdp illustrated that clotting time and rate (as measured by TEG) were not significantly affected at 10nM FV, less than 50% of the “normal” 23nM concentration. Clot strength was unaffected by FV depletion. To examine the contributions of platelet FVa, isolated and washed platelets were introduced in increasing amounts to FVdp and PFP; the rate of clotting and clot strength aligned between the two plasmas given equivalent numbers of platelets, emphasizing the importance of platelet FVa. At lower platelet concentrations (particularly 10,000 to 100,000 / µl), there were statistically significant differences in clotting time between FVdp and normal plasma, but these differences diminished with increasing platelet concentration. To evaluate the resistance of the platelet fraction to APC effects, 200,000 platelets / µl were suspended in FVdp and treated with a titration of APC from 1pM to 100nM. The samples were unaffected by APC up to a dose of at least 10nM as measured by TEG clotting parameters, well above the amounts found in the trauma patients or drotrecogin alfa-treated subjects. Conclusions FV-deficiency affects clotting only in cases of severe depletion (<50%). In a low shear in vitro closed system, APC does not affect coagulation except at very high levels (>1nM). Healthy platelets in sufficient quantity are capable of rescuing FV-deficiency and are resistant to APC effects. APC is unlikely to be the main driver of ACoT. Disclosures: No relevant conflicts of interest to declare.
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Lu, Xiyuan, Hong Liu, Lianguo Wang, and Saul Schaefer. "Activation of NF-κB is a critical element in the antiapoptotic effect of anesthetic preconditioning." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 5 (May 2009): H1296—H1304. http://dx.doi.org/10.1152/ajpheart.01282.2008.
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Anesthetic preconditioning (APC), defined as brief exposure to inhalational anesthetics before cardiac ischemia-reperfusion (I/R), limits injury in both animal models and in humans. APC can result in the production of reactive oxygen species (ROS), and prior work has shown that APC can modify activation of NF-κB during I/R, with consequent reduction in the expression of inflammatory mediators. However, the role of NF-κB activation before I/R is unknown. Therefore, these experiments tested the hypothesis that APC-induced ROS results in activation of NF-κB before I/R, with consequent increased expression of antiapoptotic proteins such as Bcl-2 and decreased apoptosis. Experiments utilized an established perfused heart rat model of sevoflurane APC and I/R. The role of NF-κB was defined by a novel method of transient inhibition of the regulatory kinase IKK using the reversible inhibitor SC-514. In addition to functional measures of left ventricular developed and end-diastolic pressure, phosphorylation of IκBα and activation of NF-κB were measured along with cytosolic protein content of Bcl-2, release of cytochrome c, and degradation of caspase-3. APC resulted in ROS-dependent phosphorylation of IκBα and activation of NF-κB before I/R. APC also increased the expression of Bcl-2 before I/R. In addition to functional protection following I/R, APC resulted in lower release of cytochrome c and caspase-3 degradation. These protective effects of APC were abolished by transient inhibition of IκBα phosphorylation and NF-κB activation by SC-514 followed by washout. ROS-dependent activation of NF-κB by APC before I/R is a critical element in the protective effect of APC. APC reduces apoptosis and functional impairment by increasing Bcl-2 expression before I/R. Interventions that increase NF-κB activation before I/R should protect hearts from I/R injury.
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Madhusudhan, Thati, Hongjie Wang, Sandra Müller-Krebs, Vedat Schwenger, Martin G. Zeier, Peter Nawroth, Charles Esmon, and Berend Isermann. "PAR-3 Activation by Activated Protein C: a Novel Podocyte Protective Signalling Pathway." Blood 116, no. 21 (November 19, 2010): 329. http://dx.doi.org/10.1182/blood.v116.21.329.329.
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Abstract Abstract 329 Activated protein C (APC) has anti-coagulant and cytoprotective effects. Recently we have shown that APC protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. The cytoprotecive effects of APC in endothelial cells require endothelial protein C receptor (EPCR) and protease activated recetor-1 (PAR-1). However, the signalling mechanism through which APC exerts its cytoprotective effects in podocytes is not known. Here we have used a mouse model of LPS-mediated podocyte injury and show that APC protects against LPS induced podocyte apoptosis and proteinuria. These cytoprotective effects of APC require activation of PAR-3. Supplementary in vitro experiments were performed using immortalized differentiated human and mouse podocytes to delineate the receptor mechanism. LPS administration (200 μg/mice, i.p.) induced podocyte apoptosis, foot process effacement and albuminuria by 24h. Administration of APC (20 μg/ mice, i.v, 6 h after LPS injection) protected against podocyte apoptosis, foot process effacement and albuminuria. Pre-incubation of APC with HAAPC antibody which blocks the anticoagulant, but not the cytoprotective properties of APC did not abolish the cytoprotective effect, establishing that the protective effect of APC is independent of its anticoagulant property. To demonstrate the direct cytoprotective effect of APC on podocytes, FITC labelled APC was administered i.v. Within 3 min FITC-APC accumulated in the pericapillary space of mouse glomeruli. In addition significantly elevated levels of PC were detected in urine samples of LPS treated mice as well as of diabetic patients or mice. These results suggest that APC transverses the glomerular filtration barrier, thus being able to directly modulate podocyte signalling in vivo. Furthermore experiments with PAR-3-/- mice, EPCR deficient (EPCRd/d) mice or PAR-1 antagonist (pepducin PI pal12S) showed that the cytoprotective effect of APC in podocytes is independent of EPCR but requires PAR-3 and PAR-1. In vitro APC (2 nM) inhibited puromycin aminonucleoside (PAN) induced podocyte apoptosis. These anti-apoptotic effect of APC require limited proteolysis of PAR-3 and cross-activation of either PAR-1 (mouse, rat podocytes) or PAR-2 (human podocytes). In addition, ectopic expression of PAR-3 in mesangial cells which lack PAR-3 is sufficient to render these cells APC sensitive and inhibit staurosporine induced apoptosis. In conclusion, we demonstrate a novel cyto-protective mechamism of APC in an acute model of nephropathy, which depends on APC mediated limited proteolysis of PAR-3. This novel podocyte protective signalling pathway may lay ground to the delineation of new pathophysiological concepts and therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.
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Montalvo Corral, Maricela, Lucinda Puebla Clark, Guadalupe López Robles, Itzel Reyes Duarte, Guillermo López Cervantes, and Silvia Yolanda Moya Camarena. "Conjugated linoleic acid enhances intestinal mucosal innate immunity against parasite Giardia lamblia in a murine model." Nova Scientia 10, no. 21 (October 15, 2018): 228–46. http://dx.doi.org/10.21640/ns.v10i21.1578.
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Introduction: Giardia lamblia, a protozoan intestinal parasite is able to evade or suppress defense mechanisms, such as innate response. Antigen presenting cells (APC) like dendritic cells may orchestrate an immune response and support a more effective adaptive defense. Conjugated linoleic acid (CLA), a dietary lipid, has shown biological activity on APC. The aim was to evaluate the effect of CLA in intestinal innate response measuring the frequency of mucosal APC populations in Giardia lamblia murine infection.Method: The giardiasis infection model was established in C3H/HeN mice (n=32), and parasite load was followed at 0, 2, 6 and 8 days post infection (dpi). APC obtained from small intestine by enzymatic digestion were assessed by flow cytometry. Oral supplementation with CLA (50:50 of cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers) and placebo control begun three days before infection and continued during first week post infection.Results: Infection kinetics showed a peak of trophozoites at 6 dpi in acute phase. Parasite load was lower in CLA group in comparison with control infected group (p<0.05). Conjugated linoleic acid stimulates innate immune response, percentage of intestinal CD11chiMHC-IIhi increases after 2 dpi, meanwhile CD11chiMHC-IIhiCD103+ APC was higher after three days of CLA supplementation than control (p<0.05). Also, the CD11c+ F4/80+ population shown a percentage increases after 6 and 8 dpi by the effect of treatment and time of infection (p<0.05).Conclusion: In conclusion CLA increased percentages of small intestine APC phenotypes CD11chiMHCIIhi, CD11chiMHCIIhiCD103+ and CD11c+ F4/80+, and reduced G. lamblia parasitic load. Further studies are needed to elucidate potential mechanisms involved in CLA immunomodulatory effect and its contribution in adaptive immune response.
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Maitra, Radhashree, Thongthai Thavornwatanayong, Madhu Kumar Venkatesh, Carol Chandy, Dov Vachss, Titto Augustine, Hillary Guzik, Wade Koba, Qiang Liu, and Sanjay Goel. "Development and Characterization of a Genetic Mouse Model of KRAS Mutated Colorectal Cancer." International Journal of Molecular Sciences 20, no. 22 (November 13, 2019): 5677. http://dx.doi.org/10.3390/ijms20225677.
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Patients with KRAS mutated colorectal cancer (CRC) represent a cohort with unmet medical needs, with limited options of FDA-approved therapies. Representing 40–45% of all CRC patients, they are considered ineligible to receive anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Although several mouse models of CRC have been developed during the past decade, one genetically resembling the KRAS mutated CRC is yet to be established. In this study C57 BL/6 mice with truncated adenomatous polyposis coli (APC) floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate an APCf/f KRAS+/f mouse colony. In another set of breeding, APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after the second round of inbreeding. The final model of the disease was generated by the cross of the two parental colonies and viable APC f/f KRAS +/f CDX2-Cre-ERT2 (KPC: APC) were genotyped and characterized. The model animals were tamoxifen (TAM) induced to generate tumors. Micro-positron emission tomography (PET) scan was used to detect and measure tumor volume and standard uptake value (SUV). Hematoxylin and eosin (H&E) staining was performed to establish neoplasm and immunohistochemistry (IHC) was performed to determine histological similarities with human FFPE biopsies. The MSI/microsatellite stable (MSS) status was determined. Finally, the tumors were extensively characterized at the molecular level to establish similarities with human CRC tumors. The model KPC: APC animals are conditional mutants that developed colonic tumors upon induction with tamoxifen in a dose-dependent manner. The tumors were confirmed to be malignant within four weeks of induction by H&E staining and higher radioactive [18F] fluoro-2-deoxyglucose (FDG) uptake (SUV) in micro-PET scan. Furthermore, the tumors histologically and molecularly resembled human colorectal carcinoma. Post tumor generation, the KPC: APC animals died of cachexia and rectal bleeding. Implications: This model is an excellent preclinical platform to molecularly characterize the KRAS mutated colorectal tumors and discern appropriate therapeutic strategies to improve disease management and overall survival.
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Okonogi, Siriporn, Pimpak Phumat, Sakornrat Khongkhunthian, Pisaisit Chaijareenont, Thomas Rades, and Anette Müllertz. "Development of Self-Nanoemulsifying Drug Delivery Systems Containing 4-Allylpyrocatechol for Treatment of Oral Infections Caused by Candida albicans." Pharmaceutics 13, no. 2 (January 27, 2021): 167. http://dx.doi.org/10.3390/pharmaceutics13020167.
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Clinical use of 4-Allylpyrocatechol (APC), a potential antifungal agent from Piper betle, is limited because of its low water solubility. The current study explores the development of the self-nanoemulsifying drug delivery system (SNEDDS) containing APC (APC-SNEDDS) to enhance APC solubility. Results demonstrated that excipient type and concentration played an important role in the solubility of APC in the obtained SNEEDS. SNEDDS, comprising 20% Miglyol 812N, 30% Maisine 35-1, 40% Kolliphor RH40, and 10% absolute ethanol, provided the highest loading capacity and significantly increased water solubility of APC. Oil-in-water nanoemulsions (NE) with droplet sizes of less than 40 nm and a narrow size distribution were obtained after dispersing this APC-SNEDDS in water. The droplets had a negative zeta potential between −10 and −20 mV. The release kinetics of APC from APC-SNEDDS followed the Higuchi model. The NE containing 1.6 mg APC/mL had effective activity against Candida albicans with dose-dependent killing kinetics and was nontoxic to normal cells. The antifungal potential was similar to that of 1 mg nystatin/mL. These findings suggest that APC-SNEDDS are a useful system to enhance the apparent water solubility of APC and are a promising system for clinical treatment of oral infection caused by C. albicans.
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Lee, Margaret S., Karen A. D'Amour, and Jackie Papkoff. "A yeast model system for functional analysis of β-catenin signaling." Journal of Cell Biology 158, no. 6 (September 16, 2002): 1067–78. http://dx.doi.org/10.1083/jcb.200204063.
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We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of β-catenin (β-cat) signaling. Coexpression of mammalian β-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both β-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with β-cat, reduces β-cat binding to TCF4, and leads to increased cytoplasmic localization of β-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to β-cat and restores reporter gene transcription. Additional β-cat signaling components, Axin and glycogen synthase kinase 3β, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein β-transducin repeat-containing protein reduces the stability of β-cat and decreases reporter activation. Thus, we have reconstituted a functional β-cat signal transduction pathway in yeast and show that β-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.
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Lee, Chang Moo, Hyun Tae Joo, and Je Sun Han. "Forecasting Long-term Housing Demand based on APC Model." Korean Association for Housing Policy Studies 25, no. 1 (February 28, 2017): 5–34. http://dx.doi.org/10.24957/hsr.2017.25.1.05.
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Stoddart, Angela, Jianghong Wang, Chunmei Hu, Anthony A. Fernald, Elizabeth M. Davis, Jason X. Cheng, and Michelle M. Le Beau. "Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apcdel/+ MDS mouse model." Blood 129, no. 22 (June 1, 2017): 2959–70. http://dx.doi.org/10.1182/blood-2016-08-736454.
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Key Points Loss of 1 copy of Ctnnb1 (encoding β-catenin) in an Apc-haploinsufficient microenvironment prevents the development of MDS. Modulation of WNT signaling in the niche using pyrvinium inhibits the development of MDS in Apc-haploinsufficient mice.
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Schuettrumpf, Joerg, Alexander Schlachterman, Jianxiang Zou, Christian Furlan Freguia, Stefano Baila, and Valder R. Arruda. "Assessing the Role of Continuous Activated Protein C (APC) Expression in Murine Thrombosis Models." Blood 106, no. 11 (November 16, 2005): 1030. http://dx.doi.org/10.1182/blood.v106.11.1030.1030.
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Abstract The protein C (PC) pathway plays a major role in the interface between coagulation and inflammation. APC has both anticoagulant and anti-inflammatory properties and is the only effective treatment in patients with severe sepsis. However, assessment of APC’s therapeutic effect on other complex disease models has been compromised by its short half-life (15 min) and by difficulties in monitoring protein levels. To overcome this limitation we used adeno-associated viral (AAV) vectors encoding the PC zymogen or APC for hepatocyte specific gene expression. For direct APC secretion we introduced an extra cleavage site adjacent to the activation peptide for the intracellular protease PACE/furin. Three dose cohorts of C57Bl/6 mice (n=4–6 per group) were injected for either AAV-APC or AAV-PC. A single vector injection resulted in continuous sustained long-term PC or APC expression without signs of liver toxicity. APC functional activity was restricted to AAV-APC-treated mice in which APC plateau levels of 88±43, 162±48, or 263±64 ng/ml were determined in a dose dependent manner. Further, AAV-APC expression consisted mainly of APC because no PC was detected by a zymogen specific ELISA. Only APC expressing mice presented enhanced anticoagulation as determined by 11 to 41 % prolongation of the aPTT values (p<0.05–0.005) and decreased thrombin/antithrombin III complex (TAT) levels (from 30 at baseline to 20, 14, or 12 ng/ml, p<0.05–0.0005). Next, we tested whether APC or PC would provide protection against vascular injury at both micro- and macrocirculation levels of living animals. No thrombus formation was detected in APC expressing mice (n=4) following FeCl3-injury of the carotid artery in contrast to uninjected or PC expressing controls (7 thrombi in 7 mice, p<0.01). Anticoagulant efficacy was then evaluated by real-time imaging of thrombus formation following laser induced arteriole injury using widefield intravital microscopy. In AAV-APC treated mice we observed dose dependent anticoagulation: 8 thrombi /12 injury sites in mice expressing ~80 ng/ml, 3/10 at ~160 ng, and 1/7 at ~260 ng/ml APC compared to 42/42 in untreated controls (P<0.001-0.0001). Expression of PC resulted in prevention of thrombus formation only at the highest expression levels of 4000 ng/ml (5/7, p<0.02) but not at 2000 ng/ml (10/10). When these animals were challenged by tail clipping, blood loss was increased only for mice with the highest APC levels by 2-fold (p<0.05). Moreover, at all levels of APC no changes in wound healing rates were observed following punch biopsy. Treatment of homozygous mice for the factor V Leiden (FVL) mutation with the same vector doses (n=3/group) resulted in a similar anticoagulant effect based on the aPTT with 18–27 % prolongation (p<0.05), or based on TAT-levels, dropping from 56.9 ng/ml at baseline to 28.1, 12.9, or 8.0 ng/ml( p<0.05–0.0005). This data shows that continuous expression of APC can overcome the inherited proteolytic resistance of FVL to APC. In summary, these results demonstrate that APC levels, within the range already obtained in humans by protein infusion (up to 400 ng/ml), provide antithrombotic activity dependent on the injury and/or vessel size. In our model, human APC levels of 160 ng/ml present effective anticoagulant effect without increasing the risk of bleeding. This strategy ensures easy assessment of the role of APC in complex disease models at closely defined circulating levels.
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van den Boom, Frans M., and Kees de Rijk. "The Schorerstichting Model AIDS Program." AIDS Patient Care 4, no. 6 (December 1990): 41–46. http://dx.doi.org/10.1089/apc.1990.4.41.
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Zhang, Ziguo, Jing Yang, Eric H. Kong, William C. H. Chao, Edward P. Morris, Paula C. A. da Fonseca, and David Barford. "Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)." Biochemical Journal 449, no. 2 (December 14, 2012): 365–71. http://dx.doi.org/10.1042/bj20121374.
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Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.
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Mizutani, Akio, Kenji Okajima, Mitsuhiro Uchiba, and Takayuki Noguchi. "Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation." Blood 95, no. 12 (June 15, 2000): 3781–87. http://dx.doi.org/10.1182/blood.v95.12.3781.
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Abstract We examined whether activated protein C (APC) reduces ischemia/reperfusion (I/R)–induced renal injury by inhibiting leukocyte activation. In a rat model, intravenous administration of APC markedly reduced I/R-induced renal dysfunction and histological changes, whereas intravenous administration of dansyl glutamylglycylarginyl chloromethyl ketone–treated factor Xa (DEGR-FXa; active-site–blocked factor Xa), heparin or diisopropyl fluorophosphate–treated APC (DIP-APC; inactive derivative of ARC) had no effect. Furthermore, APC significantly inhibited the I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability, whereas neither DEGR-FXa, heparin, nor DIP-APC produced such effects. Renal I/R-induced increases in plasma levels of fibrin degradation products were significantly inhibited by APC, DEGR-FXa, and heparin. These observations suggest that APC reduces I/R-induced renal injury independently of its anticoagulant effects but in a manner dependent on its serine protease activity. Renal levels of tumor necrosis factor- (TNF-), rat interleukin-8, and myeloperoxidase were significantly increased after renal I/R. These increases were significantly inhibited by APC but not by DEGR-FXa, heparin, or DIP-APC. Leukocytopenia produced effects similar to those of APC. These findings strongly suggest that APC protects against I/R-induced renal injury not by inhibiting coagulation abnormalities but by inhibiting activation of leukocytes that play an important role in I/R-induced renal injury. Inhibition of leukocyte activation by APC could be explained by the inhibitory activity of TNF-.
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Mizutani, Akio, Kenji Okajima, Mitsuhiro Uchiba, and Takayuki Noguchi. "Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation." Blood 95, no. 12 (June 15, 2000): 3781–87. http://dx.doi.org/10.1182/blood.v95.12.3781.012k18_3781_3787.
Full textAbstract:
We examined whether activated protein C (APC) reduces ischemia/reperfusion (I/R)–induced renal injury by inhibiting leukocyte activation. In a rat model, intravenous administration of APC markedly reduced I/R-induced renal dysfunction and histological changes, whereas intravenous administration of dansyl glutamylglycylarginyl chloromethyl ketone–treated factor Xa (DEGR-FXa; active-site–blocked factor Xa), heparin or diisopropyl fluorophosphate–treated APC (DIP-APC; inactive derivative of ARC) had no effect. Furthermore, APC significantly inhibited the I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability, whereas neither DEGR-FXa, heparin, nor DIP-APC produced such effects. Renal I/R-induced increases in plasma levels of fibrin degradation products were significantly inhibited by APC, DEGR-FXa, and heparin. These observations suggest that APC reduces I/R-induced renal injury independently of its anticoagulant effects but in a manner dependent on its serine protease activity. Renal levels of tumor necrosis factor- (TNF-), rat interleukin-8, and myeloperoxidase were significantly increased after renal I/R. These increases were significantly inhibited by APC but not by DEGR-FXa, heparin, or DIP-APC. Leukocytopenia produced effects similar to those of APC. These findings strongly suggest that APC protects against I/R-induced renal injury not by inhibiting coagulation abnormalities but by inhibiting activation of leukocytes that play an important role in I/R-induced renal injury. Inhibition of leukocyte activation by APC could be explained by the inhibitory activity of TNF-.
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Griffin, John H., Berislav V. Zlokovic, and Laurent O. Mosnier. "Activated protein C, protease activated receptor 1, and neuroprotection." Blood 132, no. 2 (July 12, 2018): 159–69. http://dx.doi.org/10.1182/blood-2018-02-769026.
Full textAbstract:
Abstract Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role as a plasma protease, pharmacologic APC is a pleiotropic protease that activates diverse homeostatic cell signaling pathways via multiple receptors on many cells. Engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks >90% anticoagulant activity but retains normal cell signaling activities. This 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein–coupled receptor, protease activated receptor 1. Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC–induced signaling that arises due to APC’s cleavage in protease activated receptor 1 at a noncanonical Arg46 site. This cleavage causes biased signaling that provides a major explanation for APC’s in vivo mechanism of action for neuroprotective activities. 3K3A-APC appeared to be safe in ischemic stroke patients and reduced bleeding in the brain after tissue plasminogen activator therapy in a recent phase 2 clinical trial. Hence, it merits further clinical testing for its efficacy in ischemic stroke patients. Recent studies using human fetal neural stem and progenitor cells show that 3K3A-APC promotes neurogenesis in vitro as well as in vivo in the murine middle cerebral artery occlusion stroke model. These recent advances should encourage translational research centered on signaling selective APC’s for both single-agent therapies and multiagent combination therapies for ischemic stroke and other neuropathologies.
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Joyce, David E., Haifeng Xu, Sumeet Mathur, and Francis J. Castellino. "Performance of Murine Soluble Thrombomodulin (rmsTM) in a Murine Model of Endotoxin-Induced Sepsis." Blood 108, no. 11 (November 16, 2006): 3933. http://dx.doi.org/10.1182/blood.v108.11.3933.3933.
Full textAbstract:
Abstract The endothelial surface protein thrombomodulin (TM) generates activated protein C (aPC) that protects from sepsis-related DIC, vascular inflammation, and apoptosis. The purpose of this study is to evaluate a novel recombinant mouse soluble TM (Eli Lilly, Indianapolis, IN) in a murine lethal endotoxin (LPS) sepsis model to determine both kinetic and survival endpoints. A dose effective in rodent reperfusion studies was employed in this sepsis model (D. Berg, Eli Lilly). C57BL6 mice were injected concomitantly with rmsTM 5 mg/kg (s.c.) and LPS 14 ug/g (i.p.) at time zero. Plasma concentrations were determined from citrated plasma at 3, 12, 24, and 48 hr, and plasma rmsTM concentrations were determined by ELISA. In vitro determinations of mouse aPC amidolytic activity and rmsTM dependent fibrinogen clotting time assays utilized rmsTM, murine thrombin, fibrinogen, and Protein C (Enzyme Research Labs). The in-vivo time-dependent mouse plasma kinetic studies confirmed plasma concentrations comparable to that achieved in human sTM antithrombotic studies. Thus, an rmsTM dose of 5 mg/kg was chosen for the 7-day LPS survival study (20% survival model). Twenty age-and weight-matched male C57BL6 mice, age 8–14 weeks, were treated with LPS 14 ug/g or LPS and rmsTM, 5 mg/kg, s.c. Survival curves were analyzed by Wilcoxon rank sum test (power 80%, alpha 2.5, p<0.05). An initial time course after rmsTM s.c. injection yielded a peak plasma rmsTM level at 3 hr (5,000 ng/mL) which persisted up to 48 hr (2,000 ng/mL). In vitro activation of aPC was saturating (2,000 ng/mL aPC with <20 ng/mL rmsTM). Prolongation of the thrombin clot time occurred at approximately 10 ug/ml of rmsTM. The LPS survival study in C57BL6 administered LPS (14 ug/g, i.p.) or LPS and rmsTM 5mg/kg s.c. demonstrated no survival difference (P=0.63). In vitro evaluation of aPC generation by rmsTM yielded ample amounts of aPC. Similarly, reduction of thrombin activity by rmsTM occurred at higher rmsTM concentrations. Thus, the lack of survival difference seen between endotoxemic mice treated with saline or adjunctive rmsTM in this model may be from the lack of thrombin inhibition, the lack of persistent (rmsTM:Thrombin) PC activation, or by more efficient clearance of aPC. Species specificity of TM is less likely a contributing factor. Further elucidation of the murine thrombin/thrombomodulin regulatory mechanism in vivo may be necessary to help explain our unexpected sTM survival results.