Combinatorial Antimicrobicidal Peptides Exhibit Enhanced Activity Against Bacterial Contaminants In Stored Platelet Concentrates (PCs).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1124-1124
Author(s):  
Krishna Mohan V. Ketha ◽  
Shilpakala Sainath Rao ◽  
Chintamani D Atreya
Keyword(s):  
Conflicts Of Interest ◽  
Microbial Reduction ◽  
Transfusion Medicine ◽  
Antimicrobial Proteins ◽  
Microbicidal Activity ◽  
Spiked Sample ◽  
Bacterial Contaminants ◽  
Fold Reduction ◽  
Colony Forming Units ◽  
Health Need

Abstract Abstract 1124 Introduction: Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. Novel proof-of-concepts for microbial reduction that enhance risk-to-benefit ratio of the treated products are still an unmet public health need in transfusion medicine. We have recently shown that synthetic antimicrobicidal peptides (AMPs) originating from thrombin-induced human platelet-derived antimicrobial proteins (named PD1-PD4) and repeats of Arginine-Tryptophan (RW1-RW5), demonstrate microbicidal activity against bacteria in plasma and PCs. In the present study we selected RW2, RW3 and RW4 and tested the effect of the peptides individually and in various combinations (cocktail) to see whether the cocktail approach enhances the antimicrobicidal activity of these peptides. Methods: The RW2, RW3 and RW4 peptides were tested on platelet samples spiked with 104 colony forming units (cfu) of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. Each spiked sample was incubated individually with 10 mM of RW2, RW3, and RW4 peptides for 1 hour at 37°C to observe their individual effect. Peptides were then combined in the following way to assess their cocktail effect: RW2+RW3; RW2+RW4; RW3+RW4; RW2+RW3+RW4. Spiked sample without any peptide was included as control. Following incubation with the peptide a ten-fold dilution of the inoculum was made and a fixed volume plated on nutrient agar plates. The plates were incubated at 37°C for 18–24 hours and colonies were counted and expressed as cfu/ml. Results: Our analysis revealed that individual peptides RW2, RW3 and RW4 were either ineffective or were moderately microbicidal (100-fold reduction) against S. aureus, S. epidermidis and P. aeruginosa. However, in combination (cocktail), these peptides together demonstrated enhanced microbicidal activity against bacteria in spiked platelets. The combinations, RW2+RW4, RW3+RW4 and RW2+RW3+RW4 were the most effective that resulted in 1000-fold reduction of all three bacteria tested. Conclusion: The present study conclusively demonstrates the synergistic effect of antimicrobicidal peptides whose combined microbicidal activity is 1000-fold higher than when the peptide is acting alone. By selecting appropriate combination of AMPs against specific bacteria of interest, new proof-of-concepts could be developed that are alternatives to the current pathogen reduction agents. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures: No relevant conflicts of interest to declare.

Salmonella Surrogate Reduction Using Industrial Peanut Dry Roasting Parameters

2014 ◽  
Vol 41 (2) ◽  
pp. 72-84 ◽  
Author(s):  
D. Poirier ◽  
T. H. Sanders ◽  
J. P. Davis
Keyword(s):  
Linear Trend ◽  
Air Flow ◽  
Microbial Reduction ◽  
Case Scenario ◽  
Worst Case ◽  
Log Reduction ◽  
Color Development ◽  
Colony Forming Units ◽  
L Value ◽  
Different Origins

ABSTRACT Studies were conducted to evaluate the effectiveness of industrial peanut dry roasting parameters in Salmonella reduction using a Salmonella surrogate, Enterococcus faecium, which is slightly more heat tolerant than Salmonella. Runner-type peanuts were inoculated with E. faecium and roasted in a laboratory scale roaster simulator in which temperature, airflow, airflow direction and bed depth were highly controlled, allowing for conditions that duplicate industrial dry roasting. Temperature data were collected at the top, middle and bottom of the roasting bed in addition to internal peanut temperature via thermocouples in the bed of peanuts and embedded in a peanut. Regardless of roast conditions, peanuts in the middle of the roasting bed received the least amount of heat and hence, represent the worst case scenario for microbial reduction. E. faecium reductions, reported as the logarithm of colony forming units/g (log CFU/g), followed a linear trend with increasing roasting time when peanuts were roasted at 149, 163, and 177 C, with > 5-log CFU/g reductions occurring at the middle of the peanut bed after 21, 15 and 11 min, respectively, at a bed depth of 75 mm and an air flow of 1.3 m/s. Increased air flow increased E. faecium reduction. At 16 min roast time and a 75 mm bed depth, reduction at the middle of the bed was ≤ 3-log CFU/g at 1 m/s and > 5-log CFU/g at 1.3 m/s. When all other roast parameters were held constant, decreasing bed depth also increased reduction of E. faecium in the middle of the bed. Comparing various samples roasted at 149, 163 and 177 C over a range of times, roast color (Hunter L-value) was positively correlated (R2  =  0.73) with the log reduction of E. faecium. Most peanuts with an L-value darker than 53, a common threshold for light roast had ≥ 5-log CFU/g reductions; however, further study is required, including roasting peanuts from different origins and maturity, to fully understand the implications of roast color development and microbial reduction. This work provides valuable practical information for manufacturers of roasted peanuts when validating Salmonella reductions under a particular set of roasting parameters.

scholarly journals Safety Evaluation of Individual Pillboxes to Control Cross-Contamination in the Drug Circuit in Hospitals

2019 ◽  
Vol 16 (20) ◽  
pp. 3878
Author(s):  
Dussart Claude ◽  
Boulliat Caroline ◽  
Camal Isabelle ◽  
Bourgeois Denis ◽  
Carrouel Florence
Keyword(s):  
Potential Role ◽  
Safety Evaluation ◽  
Contamination Rate ◽  
Cross Contamination ◽  
Medical Treatments ◽  
Bacterial Contaminants ◽  
Colony Forming Units ◽  
Resistant Strains ◽  
The Military

This study aims to evaluate the potential role of pillboxes used for the preparation and delivery of individual daily medical treatments in the drug circuit of the Military Instruction Hospital (France) as reservoirs of bacterial contaminants. Samples were obtained from 32 pillboxes after decontamination (T1), after preparation in the pharmacy (T2), after use in two different medical units (T3), and again after usual mechanical washing (T4). Qualitative (identification and antibiotic susceptibility) and quantitative (contamination rate and number of colony forming units—CFUs) bacteriological tests were performed. Susceptible and resistant strains of environmental saprophytes were identified. The pillbox contamination rate was relatively low at T1 (13%). It was significantly increased at T2 (63%, p = 0.001 vs. T1), again at T3 (88%, p < 0.05 vs. T2, p < 0.001 vs. T1), and finally decreased dramatically at T4 (31%, p < 0.001 vs. T3, p > 0.05 vs. T1). The number of CFUs was significantly increased at T2 compared with that of T1 (36.7 ± 13.4 and 5.36 ± 3.64, respectively, p < 0.001) and again at T3 (84.4 ± 19.4, p < 0.001 vs. T1 and T2) and was significantly reduced at T4 (7.0 ± 2.0 vs. T3, p < 0.001) to a level that was not significantly different from that at T1. So, the use of pillboxes to deliver individual medications to patients in the hospital is a potential risk factor for bacterial cross-contamination.

Novel Antimicrobial Peptide-Based Blood Safety Strategies for Bacterial Reduction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3044-3044
Author(s):  
Ketha V. K. Mohan ◽  
Shilpakala Sainath Rao ◽  
Chintamani D Atreya
Keyword(s):  
Bacillus Cereus ◽  
Safety Concern ◽  
Colony Count ◽  
Transfusion Medicine ◽  
Plasma Samples ◽  
Bacterial Reduction ◽  
E Coli ◽  
Spiked Sample ◽  
Tryptophan Residues ◽  
Synthetic Antimicrobial Peptides

Abstract Bacterial contamination of blood and blood components is a major safety concern in transfusion medicine. In order to facilitate safer transfusion products to the end users, there is a critical need for novel proof-of-concept ideas for pathogen reduction, which are different from the current ones that outweigh the associated toxicity and/or contamination risk. Present study involves use of nine novel synthetic antimicrobial peptides (four originated from thrombin-induced human platelet derived antimicrobial proteins named PP1-PP4 and five having 1–5 repeats of arginine and tryptophan residues, named DP1-DP5. These peptides were tested on plasma samples spiked with 10-fold dilutions of 5 different bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus cereus) that are important to the field of transfusion medicine and analyzed whether these spiked plasma samples could be cured of the pathogens. Each spiked sample was incubated with a peptide (PP1-PP4 and DP1-DP5) for 2 hours at 37°C. Following incubation, a fixed volume of the inoculum was plated on nutrient agar plates and incubated overnight at 37°C for colony count. Spiked sample without any peptide was included as control. Results revealed that out of nine peptides tested, while DP3 and DP4 were active against all 5 organisms tested resulting in 50–100 % of inhibition of specific organisms, peptide PP4 was only active against E. coli, P. aeruginosa and Bacillus cereus resulting in a 30–100% reduction in the CFU/ml compared to the controls. Table 1 Organism Colony count expressed in % Control PP1 PP2 PP3 PP4 DP1 DP2 DP3 DP4 DP5 S. aureus 100 90 100 26 100 100 20 10 56 100 E. coli 100 95 100 100 71 100 93 4 18 85 P. aeruginosa 100 100 100 100 0 100 0 0 13 100 K. pneumoniae 100 100 100 100 100 74 3 0 0 25 B. cereus 100 100 100 100 50 100 100 25 77 100 Based on these results, it appears that peptides used in this study provide a new antibacterial strategy against a range of bacteria and with further studies and refinement, these peptides could prove useful towards bacterial reduction in blood and blood products. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Activated Platelet-Derived Antimicrobial Peptides Exhibit Virucidal Properties against Vaccinia Virus in Plasma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2118-2118
Author(s):  
Krishna Mohan V. Ketha ◽  
Shilpakala Sainath Rao ◽  
Chintamani D Atreya
Keyword(s):  
Antimicrobial Peptides ◽  
Vaccinia Virus ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Human Platelet ◽  
Transfusion Medicine ◽  
Spiked Sample ◽  
Log Reduction ◽  
Virus Contamination ◽  
Viral Titers

Abstract Abstract 2118 Poster Board II-95 Introduction: Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. While there has been a tremendous success in inactivating virus contamination in blood products through UV-irradiation, new and novel proof-of-concepts for microbial reduction that enhance risk to benefit ratio of the treated products are still a public health need in transfusion medicine. In the present study, we tested four novel synthetic antimicrobial peptides originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 against an enveloped virus, Vaccinia Virus (VV) spiked in plasma as a model system. We have recently shown these peptides to be useful in reducing bacterial burden in plasma and platelets. These short synthetic peptides are human platelet-derived and known to cause no immune response in humans and non-hemolytic in nature as reported by others. Methods: The PD1-PD4 peptides were tested on plasma samples spiked with 10-fold dilutions of the wild-type lab strain of Vaccinia Virus (WR strain). Each spiked sample was pre-incubated individually with a peptide (PD1-PD4) for 1 hour at 37°C. Spiked sample without any peptide was included as control. A cell culture-based standard plaque reduction assay method was utilized to monitor the virucidal effectiveness of the peptides. Minimal inhibitory concentration of the peptides was also estimated by testing the peptides at doubling dilutions of 100 μg/ml, 50 μg/ml, 25 μg/ml and 12.5 μg/ml concentrations. Results: Our analysis revealed that peptides PD3 and PD4 were potent against vaccinia virus resulting in reduction of viral titers in the plasma. PD3 peptide demonstrated the highest virucidal activity by bringing about a 2-log reduction of VV titers. PD4 peptide treatment resulted in a 1-1.5 log reduction in viral titers. The minimal inhibitory concentration analysis revealed that at 50 μg/ml concentration both the PD3 and PD4 were able to bring about a log reduction in viral titers. Conclusion: The present study reports a novel antiviral agent for reducing vaccinia virus contamination in plasma. Safety profiles of these peptides as reported by others in conjunction with our current studies, provide a new proof-of-concept that could be useful as safer and simpler alternatives to the viral reduction agents in transfusion medicine. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures: No relevant conflicts of interest to declare.

Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3669-3669
Author(s):  
Stephan Emmrich ◽  
Franziska Schmidt ◽  
Ramesh Chandra Pandey ◽  
Aliaksandra Maroz ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Gene Set Enrichment Analysis ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Lncrna Expression ◽  
Fold Reduction ◽  
Non Coding Rnas

Abstract Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs. The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis. Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs. Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA). For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays. Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluating the effectiveness of cleaning with detergent soap alone verses detergent soap followed by sanitizer on reducing aerobic microorganism numbers that are present on food contact surfaces

2018 ◽  
Author(s):  
Vincent Man ◽  
BCIT School of Health Sciences, Environmental Health ◽  
Helen Heacock
Keyword(s):  
Research Study ◽  
Microbial Reduction ◽  
P Value ◽  
Cross Contamination ◽  
Food Contact ◽  
Food Contact Surfaces ◽  
Colony Forming Units ◽  
Contact Surfaces ◽  
Alfalfa Sprouts ◽  
Chlorine Bleach

  Background: Cross-contamination is one of the leading causes of foodborne illness which poses a massive burden to an individual’s health and to the healthcare system. One way to prevent cross-contamination is through the elimination of pathogens from surfaces by properly washing with a detergent soap followed by sanitizing with a sanitizer. However, as found from a previous research study, not all restaurants in British Columbia wash and sanitize their food contact surfaces. Thus, this study aims to compare the cleaning effectiveness between using detergent soap alone verses using detergent soap followed by sanitizer. Methods: Aerobic organisms were introduced to a cutting board by cutting alfalfa sprouts and then the surface was cleaned with Dawn Detergent soap and sanitized with 200ppm of chlorine bleach sanitizing solution. 3M™ Quick Swabs were used to sample the aerobic organisms (colony forming units) prior to and after each method of cleaning. The swabs were then transferred to 3M™ Petrifilm Plates, incubated at room temperature for 4 days, and then enumerated. Results: The results show that there is a statistically significant greater microbial reduction through cleaning with detergent soap followed by sanitizer (mean log microbial reduction of 4.10) as compared to cleaning with detergent soap alone (mean log microbial reduction of 3.53). The p-value obtained is 0.003843 when α=0.05. The power was determined to be 92%. Conclusions: This study was able to conclude that cleaning with detergent soap followed by sanitizer is 0.57 log (mean log microbial reduction of 4.10 - mean log microbial reduction of 3.53) more effective at cleaning than using detergent soap alone. However, the specific log microbial reduction value for the detergent soap followed by sanitizer achieved in this study is lower than what is found in the previous studies (Gilbert, 1970; Sores et al., 2012; Rossvoll et al., 2015). A possible reason for this discrepancy may be due to the presence of soil and food debris on the surface which may have had interfered with the sanitizing ability of the chlorine bleach (Lee et al., 2007).  

scholarly journals Minimum irradiance required to inhibit microbial growth to prevent ventilatorassociated pneumonia by an endotracheal tube equipped with blue LEDs

2021 ◽  
Vol 7 (2) ◽  
pp. 239-242
Author(s):  
Ben Sicks ◽  
Christina Stock ◽  
Sarah Peter ◽  
Tobias Meurle ◽  
Katharina Hoenes ◽  
...  
Keyword(s):  
Endotracheal Tube ◽  
Artificial Respiration ◽  
Antimicrobial Properties ◽  
Glass Tube ◽  
Microbial Reduction ◽  
Microbial Pathogens ◽  
Cellular Damage ◽  
Log Reduction ◽  
Colony Forming Units ◽  
Blue Wavelength

Abstract Artificial respiration is saving lives especially in the COVID-19 pandemic, but it also carries the risk to cause ventilator-associated pneumonia (VAP). VAP is one of the most common and severe nosocomial infections, often leading to death and adding a major economic burden to the healthcare system. To prevent a proliferation of microbial pathogens that cause VAP, an endotracheal tube (ETT) equipped with blue LEDs (LED-ETT) was developed. This blue wavelength exhibits antimicrobial properties but may also harm human tracheal cells at higher irradiances. Therefore, the aim of this study was to find the minimal required irradiance for microbial reduction of 1 log level in 24 h by applying LED-ETTs. A LED-ETT with 48 blue LEDs (450 nm) was fixed in a glass tube, which served as a trachea model. The investigation was carried out with irradiations of 4.2, 6.6 and 13.4 mW/cm² at 37 °C for 24 h. The experiments were performed with Acinetobacter kookii as a surrogate of Acinetobacter baumannii, which is classified as critical by the WHO. Samples of A. kookii suspensions were taken every 4 h during irradiation from the trachea model. Bacteria concentrations were quantified by determining colony forming units (CFU)/ml. A homogeneous irradiance of only 4.2 mW/cm² generated by the blue LEDs, at a LED forward current of 3.125 mA, is sufficient to achieve a 1 log reduction of A. kookii within 24 h. The total irradiation dose within this period was 360 J/cm2. Human cells survive this dose without cellular damage. Previous studies revealed that the pathogen A. baumannii is even more sensitive to blue light than A. kookii. Therefore, blue LED-ETTs are expected to reduce A. baumannii without harming human tracheal cells.

The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1434-1434
Author(s):  
Jianhui Wang ◽  
Ruimin Pan ◽  
Michael A. Nardi ◽  
Zongdong Li
Keyword(s):  
Nadph Oxidase ◽  
In Vitro Culture ◽  
Conflicts Of Interest ◽  
Signal Pathways ◽  
Mouse Bone Marrow ◽  
Colony Forming Units ◽  
Sequential Activation ◽  
12 Lipoxygenase ◽  
Hiv 1

Abstract Abstract 1434 We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures: No relevant conflicts of interest to declare.

Exosite Interactions in Factor IX Activation by Factor XIa

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2235-2235
Author(s):  
Yipeng Geng ◽  
Ingrid M. Verhamme ◽  
Stephen B. Smith ◽  
Amanda S. Messer ◽  
Mao-fu Sun ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Heavy Chain ◽  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Fold Reduction ◽  
Factor Xia ◽  
A Site

Abstract Abstract 2235 Conversion of factor IX (fIX) to the protease factor IXaβ (fIXaβ) is an important reaction during thrombin generation at a site of vascular injury. The physiologic activators of fIX are the proteases factor VIIa and factor XIa (fXIa). The zymogen of fXIa, fXI, is a 160 kDa dimer of two identical subunits linked by a disulfide bond. Each subunit has four apple domains at the N terminus (A1-A4), and a trypsin-like catalytic domain at the C-terminus. Conversion of fXI to fXIa involves cleavage of each subunit at the Arg369-Ile370 bond, generating a heavy chain (the apple domains) and an activated catalytic domain that remains connected to the heavy chain by a disulfide bond. FXIa activates fIX in the presence of calcium ions by sequential cleavage after Arg145 (forming the inactive intermediate fIXα) and then after Arg180 to form fIXaβ. Previously, we showed that an exosite (a site on fXIa distinct from the active site) on the A3 domain of the fXIa heavy chain is a major determinant of affinity and specificity for fIX activation by fXIa (J Biol Chem 1999;274:36373 and 2005;280:23523). Evidence has also been presented for a second fIX-binding exosite on the fXIa catalytic domain. While the catalytic efficiency (kcat/Km) for fIX activation by an isolated fXIa catalytic domain (fXIaCD – no heavy chain) was ∼500 fold lower than activation by fXIa, this was reported to be due to a decrease in kcat, rather than the expected increase in Km that should accompany loss of the A3 exosite (Biochemistry 2007;46:9830). To investigate this discrepancy, we used recombinant wild type fXIa (fXIaWT), fXIa missing the exosite on the A3 domain (fXIa-PKA3) or fXIaCD to activate purified fIX and fIXα. Full progress curves were generated using densitometry of Coomassie Blue stained SDS-polyacryalmide gels imaged at infrared wavelengths. The Km and kcat for cleavage by fXIaWT of fIX after Arg145 (Km 0.09 ± 0.02 μM, kcat = 7.3 ± 0.4 min−1) and fIXα after Arg180 (Km 0.12 ± 0.02 μM, kcat = 6.8 ± 0.4 min−1) are similar, and agree with published results. FXIa/PKA3 cleaved fIX after Arg145 with a significantly higher Km (>2 μM), consistent with loss of the exosite, and leading to an ∼100-fold reduction in catalytic efficiency. Because we were not able to reach saturation, it is not clear if the kcat was affected appreciably. Catalytic efficiency for cleavage after Arg180 was ∼3000-fold lower with FXIa-PKA3 than with fXIaWT, but the slow rate of cleavage precluded clearly determining if this was due to an effect on Km or kcat. These results indicate that the A3 exosite is involved in both cleavages, and loss of the exosite has a more deleterious effect on the second cleavage after Arg180 that converts fIXα to fIXaβ than the first cleavage after Arg145 that converts fIX to fIXα. This would account for the observation that there is substantial accumulation of fIXα when fIX is activated by FXIa-PKA3, but not by fXIaWT. For fIX cleavage after Arg145 by fXIaCD, Km was again markedly increased (≥ 2 μM) compared to FXIaWT, with a modest (∼3-fold) reduction in kcat resulting in reduced catalytic efficiency that is roughly similar to that for FXIa/PKA3. The catalytic efficiency of cleavage after Arg180 by fXIaCD was ∼4000 fold reduced compared to FXIa-WT. Interestingly, when calcium was removed from the reactions, cleavage of both the Arg145 and Arg180 activation sites by fXIa-WT, but not by fXIa/PKA3 or fXIaCD, were markedly impaired, indicating both cleavages are Ca2+–dependent reactions. Cumulatively, these results indicate that an exosite on the heavy chain A3 domain is largely responsible for the Ca2+-dependent affinity of fIX and fIXα for fXIa. We used surface plasmon resonance as a complementary approach to look directly at Ca2+-dependent binding of fXIa to fIX. FXIa-WT bound to immobilized fIX with Kd 48nM, in reasonable agreement with results from the kinetic analysis. Isolated fXIa heavy chain (lacking the catalytic domain) bound with similar Kd (53 nM). In contrast, fXIa/PKA3 and fXIaCD bound poorly to fIX (Kd >2 μM). Taken as a whole, the data support the hypothesis that an exosite on the fXIa A3 domain is largely responsible for affinity and specificity of the fXIa-mediated reactions converting fIX to fIXα, and fIXα to fIXaβ. While the analysis cannot rule out minor contributions of other exosites to the reactions, they do not support the premise that there is a fIX- or fIXα-binding site on the fXIa catalytic domain that contributes substantially to initial substrate binding. Disclosures: No relevant conflicts of interest to declare.

Generation of a Transgenic Mouse with Inducible Constitutively Active Stat5a,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3399-3399
Author(s):  
Harini Nivarthi ◽  
Andriy Tsyrulnyk ◽  
Wolfgang Warsch ◽  
Zhengqi Wang ◽  
Susanne Winkler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Fold Reduction ◽  
Transgenic Founder ◽  
Constitutively Active

Abstract Abstract 3399 The Jak/Stat signalling pathway is essential for survival and proliferation of haematopoietic cells. The Stat5 transcription factors (Stat5a and Stat5b) play a crucial role in the development of various lineages of the hematopoietic system. Stat5 is activated by many cytokines and growth factors that regulate hematopoiesis. Persistent Stat5 activity is frequently found in hematopoietic cancers due to aberrant tyrosine kinase signalling. We have previously characterized a constitutively activated version of Stat5 (called oncogenic Stat5; cS5F), which mimics persistent tyrosine kinase signalling and promotes multi-lineage leukaemia development in a bone marrow transplantation model. The aim of the study is to generate a novel inducible mouse model for oncogenic Stat5a using the BAC recombineering technology. A cassette expressing cS5F (with a C-terminal FLAG tag) and a reporter gene (truncated human CD2; hCD2) was cloned, sequenced and biochemically verified for persistent tyrosine phosphorylation in absence of cytokines or growth factors. The cassette is flanked by loxP sites in opposite orientation and was used to replace the original Stat5a gene in a BAC carrying the endogenous Stat3/5 locus using BAC recombineering. Therefore, the expression of the transgene is regulated by the endogenous Stat5a promoter. The transgene is in an inverted (off) orientation and can be switched into the transcribing (on) orientation by Cre activity. Pronuclear injections were performed with the linearized and purified BAC. We obtained 5 transgenic founder lines which showed germ line transmission of the transgene. The copy number of the BAC was estimated to be 2 by Southern blot analysis. The transgenic founder lines were bred with the Rosa CreERT2 mice, and the Cre activity was induced by treating the mice with 1 mg of tamoxifen every day, for 5 consecutive days. The recombination of the transgene into the ‘on' orientation in liver, lungs, kidney, spleen and bone marrow was demonstrated by Southern blotting. The expression of the hCD2 marker was detected in various hematopoietic lineages by FACS and the expression of the transgenic protein in liver was confirmed by Western blotting with anti-Stat5a and anti-FLAG antibodies. One week after induction of the transgene, all the mice induced with tamoxifen (n=6) developed atrophic thymii with nearly a 4-fold reduction in total thymocyte numbers (p=0.0003). However, the percentage of CD8+ cells was increased more than 3 fold in the thymus (p=0.0002). Moreover, there was a 2-fold reduction in the number of early hematopoietic progenitors; defined as lin−, Sca1+ and c-kit+, in the bone marrow (n=5, p=0.0217). We have established an inducible mouse model for expression of constitutively active Stat5a under the endogenous promoter. We are currently monitoring these mice for development of cancer as they provide a competent tool to study the molecular mechanisms triggered by persistent Stat5a activity, including identification of other cooperating signalling pathways that contribute to generation of cancer. Moreover, it would allow one to test interference strategies, which could lead to potential therapeutics. Disclosures: No relevant conflicts of interest to declare.

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