Journal: Blood

Article Title: Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways

doi: 10.1182/blood-2017-10-813618

Figure Lengend Snippet: PGN induces monocyte TF expression and extrinsic pathway activation in vivo and in vitro. (A-C) PGN challenge induces monocyte TF expression in vivo and priming of the extrinsic tenase protease. (A) Serial blood smears were methanol fixed, stained overnight at 4°C with biotinylated HTF-1 monoclonal anti-human TF, followed by detection with Cy3-labeled streptavidin (Jackson ImmunoResearch, West Grove, PA). Monocytes were identified morphologically. Confocal images were acquired on a Nikon Eclipse TE2000-U inverted microscope equipped with a Nikon C1 scanning head using EZ-C1 software. Representative micrographs of TF immunostaining in time-paired PGN-challenged baboons are shown (scale bar represents 20 µm). TF staining is stronger in high-dose challenged baboons (right panel) as opposed to low-dose challenge (middle panel). For comparison, no monocyte TF staining is observed on blood smears processed before the high-dose PGN challenge (left panel). (B) Time-course quantitation of TF immunostaining on serial blood smears depicted in panel A. Monocyte TF staining was quantified in at least 5 individual fields from each time point. (C) Time-course evaluation of FVIIa-AT complexes, as a marker of in vivo FVII activation, in PGN-challenged primates. Statistical analysis and representation are consistent with Figure 1. (D-F) PGN stimulation induces de novo TF expression in primary human monocytes in vitro. Time-course analysis of TF mRNA (D), surface antigen levels (E), and TF procoagulant activity (F) on monocytes stimulated with either 10 µg/mL PGN or 1 µg/mL LPS. Data are represented as mean ± SEM from 3 experiments using independent donors. Multiple comparisons were performed by 2-way repeated measures ANOVA followed by Holm-Sidak post hoc test and statistically significant changes from unstimulated (T0) controls are depicted: *P < .05; ** P < .01; ***P < .001.

Article Snippet: 14 , 36 Other coagulation protease-antithrombin (AT) complexes were measured by enzyme-linked immunosorbent assay (ELISA) after coating with affinity-purified antibodies against the protease (clone 9G3 monoclonal anti-human factor XII [FXII], clone 1A6 monoclonal anti-human FXI, 37 affinity-purified goat anti-human FVII [R&D Systems, Minneapolis, MN]) and detection with biotinylated sheep anti-human AT (Affinity Biologicals, Ancaster, ON, Canada).

Techniques: Expressing, Activation Assay, In Vivo, In Vitro, Staining, Labeling, Inverted Microscopy, Software, Immunostaining, Comparison, Quantitation Assay, Marker, Activity Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Novel mutation in coagulation factor VII (Carmel mutation): Identification and characterization

doi: 10.1002/rth2.12407

Figure Lengend Snippet: Factor VII activity and antigenicity

Article Snippet: Factor VII antigen was determined using the AssayMax human factor VII ELISA kit (Assaypro, St. Charles, MO, USA).

Techniques: Activity Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

doi: 10.3390/ijms23095090

Figure Lengend Snippet: Complete list of unique gene specific 20 base pair gRNA targeting sequences.

Article Snippet: Open reading frame plasmids for factor II (RC208589) and factor VII (RC213143) were obtained from Origene (Rockville, MD, USA).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

doi: 10.3390/ijms23095090

Figure Lengend Snippet: Lambda SAM overexpression of coagulation factors IX and X translates into secreted protein expression. ( A ) ELISA based protein quantification of transient SAM driven overexpression of coagulation factors IX and X or ( B ) Plasmid open reading frame driven overexpression of factor VII transcript variant 2 in HEK293 cells compared to untreated cells ( n = 3 independent experiments. p values were calculated using Student’s unpaired, two-sided t test to compare treated and untreated cells (n.s. p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: Open reading frame plasmids for factor II (RC208589) and factor VII (RC213143) were obtained from Origene (Rockville, MD, USA).

Techniques: Over Expression, Coagulation, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production

doi: 10.3390/ijms23095090

Figure Lengend Snippet: Complete list of qRT-PCR probes used for detection of transcripts of interest.

Article Snippet: Open reading frame plasmids for factor II (RC208589) and factor VII (RC213143) were obtained from Origene (Rockville, MD, USA).

Techniques:

Journal: Chinese Medicine

Article Title: iTRAQ-based proteomic analysis to identify the molecular mechanism of Zhibai Dihuang Granule in the Yin-deficiency-heat syndrome rats

doi: 10.1186/s13020-017-0160-y

Figure Lengend Snippet: Verification of the differentially expressed proteins in coagulation and complement cascades by ELISA. Proteins expression were measured in the control group (n = 24), the YDH syndrome group (n = 20) and the ZDG treated group (n = 20). p values were calculated with the Mann–Whitney U-test, * p < 0.05, ** p < 0.01, *** p < 0.001. CG control group, YDHG YDH syndrome group, ZDGG ZDG treated group

Article Snippet: Rat coagulation factor VII (F7) ELISA kit (Cusabio, detection limit 0.195 ng/mL), rat fibrinogen alpha chain (Fga) ELISA kit (Cusabio, detection limit 0.025 μg/mL), rat fibrinogen beta chain (Fgb/Ab1-181/Ab1-216/Ac1-581) ELISA kit (Cusabio, detection limit 0.156 μg/mL), and rat von Willebrand Factor (vWF) ELISA kit (Cusabio, detection limit 0.078 ng/mL) were proteins in the coagulation cascade.

Techniques: Coagulation, Enzyme-linked Immunosorbent Assay, Expressing, Control, MANN-WHITNEY

Journal: Blood Transfusion

Article Title: Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

doi: 10.2450/2009.0081-08

Figure Lengend Snippet: SDS-PAGE and western blot analysis of recombinant FVII fusion protein. A; SDS-PAGE analysis. The recombinant fusion protein was purified using a nickel–sepharose column. Af ter purification, a single protein band was detected in 12% SDS-PAGE (Lane 2). The cell culture medium of CHO cells transfected with empty vector was also passed through a nickel–sepharose column, but no band was observed (Lane 1). B; Western blot analysis of eluted protein using anti-his-tag and anti-FVII antibodies (C). Eluted protein reacted with both antibodies, visualised by development with DAB solution.

Article Snippet: Purified protein was also detected using goat anti-human FVII antibody (R&D Systems, Minneapolis, MN, USA) with 0.75 μg/mL peroxidise-conjugated anti-goat IgG (Dako, Denmark) as the secondary antibody.

Techniques: SDS Page, Western Blot, Recombinant, Purification, Cell Culture, Transfection, Plasmid Preparation

Journal: Blood Transfusion

Article Title: Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

doi: 10.2450/2009.0081-08

Figure Lengend Snippet: Prothrombin time assay. The biological activity of the purified recombinant his-tagged FVII was evaluated by the prothrombin time test. A three-fold decrease in clotting time was observed when human FVII-depleted plasma was used in combination with human thromboplastin and the his-tagged recombinant FVII produced in this study. About a four-fold decrease in clotting time was observed when a commercially available preparation of recombinant FVII (NovoSeven), was tested. The experiment was performed in triplicate and the data are presented as mean ± SD.

Article Snippet: Purified protein was also detected using goat anti-human FVII antibody (R&D Systems, Minneapolis, MN, USA) with 0.75 μg/mL peroxidise-conjugated anti-goat IgG (Dako, Denmark) as the secondary antibody.

Techniques: Activity Assay, Purification, Recombinant, Coagulation, Produced