Journal: The Journal of Biological Chemistry

Article Title: Porphyromonas gingivalis -derived RgpA-Kgp Complex Activates the Macrophage Urokinase Plasminogen Activator System

doi: 10.1074/jbc.M115.645572

Figure Lengend Snippet: The RgpA-Kgp complex activates pro-uPA to form uPA. A, human pro-uPA activation by plasmin and RgpA-Kgp complex were compared in a kinetic chromogenic assay. Pro-uPA (50 nm) was incubated with plasmin (10 nm) or the RgpA-Kgp complex (10 nm) in the presence of the inhibitors α2-antiplasmin (α2-AP, 100 nm) or PAI-1 (100 nm). The uPA-specific substrate (0.6 mm; S2444) and absorbance was monitored for 90 min at 37 °C at 405 nm. B, comparison of the dose-dependent activation of pro-uPA (50 nm) by plasmin and the RgpA-Kgp complex (25 to 0.1 nm). The molar ratios measured were 500, 250, 50, 25, 10, 5, and 2 of pro-uPA to 1 of plasmin or RgpA-Kgp. The data are plotted on a log2 scale. Absorbance at 405 nm was measured after 60 min at 37 °C (n = 5, ± S.D.).

Article Snippet: Reagents The reagents were as follows: recombinant human macrophage-colony stimulating factor (M-CSF) (Chiron, Emeryville, CA); human pro-uPA was a kind gift from Novo Nordisk A/S (Copenhagen, Denmark); human glu-plasminogen (Enzyme Research Lab, South Bend, IN); human plasmin (Calbiochem), uPA and PAI-1 (Molecular Innovations, Novi, MI), and α2-antiplasmin (Innovative Research, Novi, MI); anti-human uPA mAb (clone U-16; Thermo Scientific, Waltham, MA); anti-mouse uPA mAb (clone mU1, Finsen Laboratory, Copenhagen, Denmark) ( 33 ); and mouse IgG1 (MOPC-21; BioXcell, West Lebanon, NH).

Techniques: Activation Assay, Chromogenic Assay, Incubation

Journal: bioRxiv

Article Title: Harnessing micrometer-scale tPA beads for high plasmin generation and accelerated fibrinolysis

doi: 10.1101/2024.11.06.621942

Figure Lengend Snippet: Fibrin gels were formed with ( A ) normal pooled plasma, tissue factor (1.25 pM) and CaCl 2 (8.3 mM), ( B ) fibrinogen (2 mg/mL), glu-plasminogen (10 μM), thrombin (2 U/mL), ( C ) fibrinogen (2 mg/mL), glu-plasminogen (10 μM), thrombin (2 U/mL), α2-antiplasmin (1 μM) with free tPA or tPA-beads at 37°C. Fibrinolysis was measured by absorbance (405 nm) for tPA-beads (2.25 mU/mL) with diameters of 0.1 μm (3.55×10 10 beads/mL) and 1.0 μm (1.69×10 8 beads/mL) or free tPA (2.25 mU/mL). Each line and shaded region represent the average and standard deviation of n=3.

Article Snippet: Human fibrinogen depleted of plasminogen, von Willebrand Factor and fibronectin (FIB3), human glu-plasminogen (HPG 2001) and α2-antiplasmin (α2AP) was obtained from Enzyme Research Laboratories (South Bend, IN).

Techniques: Standard Deviation

Journal: Blood Advances

Article Title: Inhibition of contact-mediated activation of factor XI protects baboons against S aureus –induced organ damage and death

doi: 10.1182/bloodadvances.2018029983

Figure Lengend Snippet: Pretreatment with 3G3 antibody prevented fibrinogen consumption, tissue fibrin deposition, and fibrinolysis activation following a lethal dose of heat-inactivated S aureus injection in baboons. (A) Time course changes of fibrinogen levels. (B) Double immunostaining for fibrin (green) and platelet marker CD41 (red) in the lung and kidney of baboons challenged with LDSA (left) or with anti FXI antibody treatment (LDSA + 3G3; right). Nuclear counterstaining is shown in blue. Confocal images were captured on a Nikon Eclipse TE2000-U inverted microscope equipped with a Nikon C1 scanning head. Images were acquired and processed using EZ-C1 software (v 3.80; Nikon, Melville NY). Scale bars, 100 µm. (C-F) Time course changes of plasma markers of fibrinolysis: t-PA (C), PAI-1 (D), plasmin-antiplasmin complexes (E), and D-dimer (F). *P < .05, **P < .01, ***P < .001.

Article Snippet: For plasmin-antiplasmin complexes, we used affinity purified goat anti-human plasminogen (2 µg/mL, Affinity Biologicals) as capture and horseradish peroxidase–conjugated goat anti-human α2-antiplasmin (1 µg/mL, Affinity Biologicals) as detection antibodies.

Techniques: Activation Assay, Injection, Double Immunostaining, Marker, Inverted Microscopy, Software

Journal: PLoS ONE

Article Title: Requirement of Plasminogen Binding to Its Cell-Surface Receptor α-Enolase for Efficient Regeneration of Normal and Dystrophic Skeletal Muscle

doi: 10.1371/journal.pone.0050477

Figure Lengend Snippet: A . Immunocytochemical staining for eMHC of MPCs in DM in the presence of MAb11G1, EACA and α2-antiplasmin. B . Quantification of the Differentiation ratio (n° eMHC positive cells/total cells). C . Quantification of the Fusion ratio (n° eMHC positive cells fused in a myotube >4 nuclei/total cells) or Myogenic Index. are mean +/− SEM (error bars). ** P <0.005. Experiments were performed in triplicates.

Article Snippet: Monoclonal antibodies MAb11G1 and MAb7H8 against α-enolase, produced in our laboratory ; ε-aminocaproic acid (EACA), Sigma; α2-antiplasmin, Loxo GmbH.

Techniques: Staining