Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of uPA with human plasma α2-macroglobulin (α2M), but not with human recombinant suPAR, results in protection from inactivation by PAI-1. scuPA (50 nM) was incubated with 150 nM suPAR, and 50 nM scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) were incubated with 2.0 μM α2M from human plasma in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C for 7 h. At the end of incubation, complexes of uPA with suPAR or α2M were isolated from each reaction mixture by IP with 20 μg of MAb ATN-658 (ATN-658 does not affect amidolytic activity of uPA complexed with suPAR) and with 20 μg of polyclonal sheep anti-human α2M antibodies. Reaction mixtures were incubated with 40 μl of 1:1 slurry of protein A/G Plus Agarose for 2 h at room temperature on a rocker. Reaction mixtures were centrifuged at 3,000–6,000 rpm using an AccuSpin Micro R centrifuge. The resin was washed 3 times with 0.6 ml of cold HEPES buffer, and 50 μl of the resin suspension (total vol 200 μl) was transferred to 96-well plates. Amidolytic uPA activity in precipitated samples was measured with (white bars) or without (gray bars) 80 nM PAI-1. The composition of each reaction mixture, antibodies used for IP, and presence of PAI-1 are shown in the table under the plot.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Clinical Proteomics, Recombinant, Isolation, Activity Assay, Suspension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of scuPA with rabbit PFs forms αM complexes possessing uPA amidolytic activity that resists PAI-1. A: IP of uPA activity by anti-rabbit α2M IgY from PFs of rabbits treated with intrapleural ED of scuPA after preincubation with 50 nM scuPA for 5 h at 37°C in 50 mM HEPES/NaOH, pH 7.4. Samples were immunoprecipitated with α2M IgY or control nonspecific IgY, and uPA amidolytic activity was measured with (white) or without (gray) added exogenous PAI-1 (80 nM) as described in materials and methods and in Fig. 5. uPA amidolytic activity was significantly (*P < 0.05) increased in both groups of PFs from rabbits IP with anti-rabbit α2M vs. those IP nonspecific IgY. B: bar plot of the uPA activity in the precipitates from PFs of animals treated with the ED of scuPA or HD of Activase (Act), Abbokinase (Abb), or vehicle (n = 2 randomly selected samples/each paired group). The precipitation was carried out with 40 μg of either anti-rabbit α2M (filled bars) or nonspecific IgY (hatched bars). The complexes were captured with 80 μl of Agarose slurry and analyzed as described in Fig. 5. C: anti-α2M but not anti-uPAR antibodies precipitate uPA activity from PFs of rabbits, treated with ED of scuPA. IP of uPA activity by polyclonal sheep anti-human α2M antibodies and anti-human uPAR MAb (cross-reacting with rabbit αM and rabbit uPAR, respectively) from PFs of rabbits treated with ED of scuPA. PFs were incubated with 40 μg of antibodies for 2 h in 50 mM HEPES/NaOH, pH 7.4, either with anti-uPAR MAb or with anti-α2M antibodies. In control experiments, PFs were supplemented with exogenous mixtures of 150 nM suPAR with either 50 nM HMW or LMW tcuPA before anti-uPAR MAb was added. Another set of controls contained PFs without addition of antibodies. The composition of each reaction mixture and antibodies used for IP are shown in the table under the plot. Complexes were precipitated with 40 μl of Protein A/G Plus Agarose, and uPA amidolytic activity was measured with (white) or without (gray) 80 nM PAI-1 added, as described in Fig. 5 legend. uPA amidolytic activity was significantly (P < 0.05) increased in PFs incubated with sheep anti-human α2M antibodies and samples supplemented with HMW tcuPA complexed with suPAR (activity without PAI-1) vs. all other samples.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Western blot analysis of α2M (A) and uPA (B) antigens in complexes immunoprecipitated from PFs of rabbits treated with intrapleural scuPA. Western blot analysis of proteins precipitated by anti-rabbit α2M (lanes 1–3) or nonspecific (lane 4) IgY from PFs of rabbits treated with ED scuPA. PFs were incubated with (lanes 2 and 4) or without (lanes 1 and 3) exogenous scuPA (50 nM) and precipitated with anti-rabbit α2M IgY as described in materials and methods and Fig. 5 legend. Samples of the resin were heated with SDS loading buffer (100°C, 2 min) and subjected to a 4–12% gradient SDS-PAGE (NuPage, Invitrogen) under nonreducing conditions. Positions of molecular weight markers (Novex, Invitrogen) are indicated to the left of the gel to allow assessment of the molecular weights of the bands. Lane 5: scuPA standard. Proteins were transferred to the PVDF membrane (Invitrogen), and membranes were incubated with anti-uPA MAb and developed with anti-human-α2M IgY-HRP conjugate (A) using ECL substrate (Pierce) and with goat anti-mouse IgG-alkaline phosphatase conjugate (B) using WesternBreeze (Invitrogen). Each set of 5 lanes (A and B) represents data obtained from the same gel.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Western Blot, Immunoprecipitation, Incubation, SDS Page, Molecular Weight, Membrane

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Rabbit α2M/uPA complexes immunoprecipitated from PFs activate plasminogen and increase uPA activity when exposed to plasmin. A: α2M complexes were isolated from PFs of animals treated with ED scuPA, high dose of Activase, Abbokinase, and with buffer saline vehicle by immunoprecipitation with anti-rabbit α2M IgY (gray) or nonspecific IgY (white). The resin was transferred into 96-well Pro-Bind plates and incubated with plasminogen (0.2 μM) and 0.2 mM chromogenic plasmin substrate (Centerchem, Switzerland) at 37°C. Plasminogen activation was determined as an increase in plasmin (PL) activity in time using SpectraMax Plus (Molecular Devices). The data are presented in the box plot format as described in Fig. 1 legend. Plasminogen activating activity was significantly increased in the PFs of rabbits treated with intrapleural scuPA (P < 0.05). B: effect of plasmin on uPA activity of α2M/uPA complexes precipitated with anti-rabbit α2M IgY from pleural fluids. PFs of animals treated with scuPA were incubated with or without a mixture of 50 nM scuPA and 100 nM PAI-1 in HEPES buffer, pH 7.4, for 5 h at 37°C. α2M/uPA complexes were precipitated with anti-rabbit IgY and Agarose with immobilized anti-IgY goat polyclonal IgG. Amidolytic uPA activity in the resin samples was measured with fluorogenic substrate Pefafluor uPA with (gray) or without (white) preincubation with 1 nM plasmin. *Significant differences (P < 0.05) between the groups.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Immunoprecipitation, Activity Assay, Isolation, Saline, Incubation, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Amidolytic urokinase plasminogen activator (uPA) activity in pleural fluids (PFs) of rabbits with tetracycline (TCN)-pleural injury treated with intrapleural single-chain (sc)uPA, Abbokinase, or Activase. Amidolytic uPA activity was measured in PFs as described in materials and methods. Activity is expressed as slopes of the linear dependencies of changes in the fluorescence emission at 440 nm (excitation 340 nm) of 50 μM fluorogenic substrate Pefafluor uPA (Centerchem) in 0.05 M HEPES/NaOH buffer pH 7.4. uPA activity was significantly increased in the pleural fluids of rabbits with TCN-induced injury treated with an effective dose (ED) of intrapleural scuPA vs. all other groups (P < 0.05), including rabbits treated with intrapleural Abbokinase [Abb; at low (LD) or high (HD) dose] or Activase (Act; LD or HD). The 0.5 ED of scuPA = 0.25 mg/ml did not effectively clear intrapleural loculation (16). S195A, inactive mutant variant of scuPA. uPA activity is indicated as arbitrary units (AU). Data are illustrated in a box plot format in which the 25 and 75% quartiles are indicated in the box, and median values are shown as horizontal lines within the box, as previously described (16).

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Activity Assay, Fluorescence, Mutagenesis, Variant Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: PFs of rabbits treated with intrapleural scuPA exhibit uPA activity that resists inhibition by plasminogen activator inhibitor-1 (PAI-1). The effects of exogenous PAI-1 on uPA amidolytic activity in the PFs of animals treated with either an effective therapeutic dose (ED) of scuPA or an ineffective dose (ED 0.5) are illustrated. Amidolytic uPA activity was determined before (gray) and after (white) addition of an excess of PAI-1 as described in materials and methods. Vehicle: PFs from PBS vehicle-treated controls for the scuPA HD- or scuPA LD-treated rabbits. Data are illustrated in the box plot format as described in Fig. 1 legend. While uPA activity in the presence of PAI-1 was lower than that without serpin in all samples, there was no statistical difference between the groups lacking PAI-1 (gray) vs. those supplemented with excess PAI-1 (white boxes, P ≥ 0.1). *Statistically significant differences (P < 0.05) between corresponding groups.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Activity Assay, Inhibition

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Time-course evidence that exogenous scuPA (A) generates uPA activity that is resistant to PAI-1, that tPA (B) does not generate PAI-1-resistant tPA activity, and that tcuPA in equivalent dose (C) can generate PAI-1-resistant uPA activity in PFs. Box plots of time dependencies of the activity of the mixtures of (A) 50 nM scuPA with PFs of a rabbit treated either with the effective dose of scuPA (gray) or saline control vehicle (white); randomly selected samples per group are n = 7 and 3, respectively. B: 50 nM single-chain tPA (sctPA; Activase) with PFs of animals treated either with high dose of Activase (gray) or saline vehicle (white); n = 5 and 3, respectively, randomly selected samples/group. C: 50 nM tcuPA added to PFs of animals treated with high dose of Abbokinase (LMW tcuPA; gray) or with buffer (white); n = 3 randomly selected samples/group. Mixtures were incubated at 37°C, in 0.05 M HEPES/NaOH, pH 7.4, for 18 h. At the end of the incubation, an excess of PAI-1 (200 nM) was added to each reaction mixture to determine the fraction of the amidolytic activity that is resistant to PAI-1. The data are illustrated using the box plot format described in Fig. 1 legend. *Significant differences (P ≤0.02) between the groups.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Activity Assay, Saline, Control, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Effect of concurrent administration of excess PAI-1 on activity of 50 nM exogenous scuPA, sctPA, or tcuPA added to PFs of control vehicle-treated animals. Box plots as described in Fig. 1 legend illustrate time dependencies of the activity of the mixtures of 100 nM PAI-1 (added to the PF immediately after the enzyme) and 50 nM exogenous sctPA (white boxes, n = 5), tcuPA (gray boxes; n = 3), and scuPA (hatched boxes; n = 4) to PFs vehicle-treated rabbits. Mixtures were incubated at 37°C in 0.05 M HEPES/NaOH, pH 7.4, for 18 h. Aliquots (6 μl) were withdrawn at the indicated times to measure the enzymatic activity using a fluorescence spectrophotometer. At the end of the incubation, an excess of PAI-1 (200 nM) was added to each reaction mixture to determine the fraction of the amidolytic activity that is resistant to PAI-1. There was a statistically significant (P ≤0.03) increase in uPA activity resistant to PAI-1 seen only in the PFs to which scuPA was added at 18 h after incubation (*).

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Activity Assay, Control, Incubation, Fluorescence, Spectrophotometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of uPA with human plasma α2-macroglobulin (α2M), but not with human recombinant suPAR, results in protection from inactivation by PAI-1. scuPA (50 nM) was incubated with 150 nM suPAR, and 50 nM scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) were incubated with 2.0 μM α2M from human plasma in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C for 7 h. At the end of incubation, complexes of uPA with suPAR or α2M were isolated from each reaction mixture by IP with 20 μg of MAb ATN-658 (ATN-658 does not affect amidolytic activity of uPA complexed with suPAR) and with 20 μg of polyclonal sheep anti-human α2M antibodies. Reaction mixtures were incubated with 40 μl of 1:1 slurry of protein A/G Plus Agarose for 2 h at room temperature on a rocker. Reaction mixtures were centrifuged at 3,000–6,000 rpm using an AccuSpin Micro R centrifuge. The resin was washed 3 times with 0.6 ml of cold HEPES buffer, and 50 μl of the resin suspension (total vol 200 μl) was transferred to 96-well plates. Amidolytic uPA activity in precipitated samples was measured with (white bars) or without (gray bars) 80 nM PAI-1. The composition of each reaction mixture, antibodies used for IP, and presence of PAI-1 are shown in the table under the plot.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Clinical Proteomics, Recombinant, Isolation, Activity Assay, Suspension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of scuPA with rabbit PFs forms αM complexes possessing uPA amidolytic activity that resists PAI-1. A: IP of uPA activity by anti-rabbit α2M IgY from PFs of rabbits treated with intrapleural ED of scuPA after preincubation with 50 nM scuPA for 5 h at 37°C in 50 mM HEPES/NaOH, pH 7.4. Samples were immunoprecipitated with α2M IgY or control nonspecific IgY, and uPA amidolytic activity was measured with (white) or without (gray) added exogenous PAI-1 (80 nM) as described in materials and methods and in Fig. 5. uPA amidolytic activity was significantly (*P < 0.05) increased in both groups of PFs from rabbits IP with anti-rabbit α2M vs. those IP nonspecific IgY. B: bar plot of the uPA activity in the precipitates from PFs of animals treated with the ED of scuPA or HD of Activase (Act), Abbokinase (Abb), or vehicle (n = 2 randomly selected samples/each paired group). The precipitation was carried out with 40 μg of either anti-rabbit α2M (filled bars) or nonspecific IgY (hatched bars). The complexes were captured with 80 μl of Agarose slurry and analyzed as described in Fig. 5. C: anti-α2M but not anti-uPAR antibodies precipitate uPA activity from PFs of rabbits, treated with ED of scuPA. IP of uPA activity by polyclonal sheep anti-human α2M antibodies and anti-human uPAR MAb (cross-reacting with rabbit αM and rabbit uPAR, respectively) from PFs of rabbits treated with ED of scuPA. PFs were incubated with 40 μg of antibodies for 2 h in 50 mM HEPES/NaOH, pH 7.4, either with anti-uPAR MAb or with anti-α2M antibodies. In control experiments, PFs were supplemented with exogenous mixtures of 150 nM suPAR with either 50 nM HMW or LMW tcuPA before anti-uPAR MAb was added. Another set of controls contained PFs without addition of antibodies. The composition of each reaction mixture and antibodies used for IP are shown in the table under the plot. Complexes were precipitated with 40 μl of Protein A/G Plus Agarose, and uPA amidolytic activity was measured with (white) or without (gray) 80 nM PAI-1 added, as described in Fig. 5 legend. uPA amidolytic activity was significantly (P < 0.05) increased in PFs incubated with sheep anti-human α2M antibodies and samples supplemented with HMW tcuPA complexed with suPAR (activity without PAI-1) vs. all other samples.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Western blot analysis of α2M (A) and uPA (B) antigens in complexes immunoprecipitated from PFs of rabbits treated with intrapleural scuPA. Western blot analysis of proteins precipitated by anti-rabbit α2M (lanes 1–3) or nonspecific (lane 4) IgY from PFs of rabbits treated with ED scuPA. PFs were incubated with (lanes 2 and 4) or without (lanes 1 and 3) exogenous scuPA (50 nM) and precipitated with anti-rabbit α2M IgY as described in materials and methods and Fig. 5 legend. Samples of the resin were heated with SDS loading buffer (100°C, 2 min) and subjected to a 4–12% gradient SDS-PAGE (NuPage, Invitrogen) under nonreducing conditions. Positions of molecular weight markers (Novex, Invitrogen) are indicated to the left of the gel to allow assessment of the molecular weights of the bands. Lane 5: scuPA standard. Proteins were transferred to the PVDF membrane (Invitrogen), and membranes were incubated with anti-uPA MAb and developed with anti-human-α2M IgY-HRP conjugate (A) using ECL substrate (Pierce) and with goat anti-mouse IgG-alkaline phosphatase conjugate (B) using WesternBreeze (Invitrogen). Each set of 5 lanes (A and B) represents data obtained from the same gel.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Western Blot, Immunoprecipitation, Incubation, SDS Page, Molecular Weight, Membrane

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Different mechanisms of interaction of PAI-1 with scuPA vs. Activase, Abbokinase, and tcuPA. Dependencies of the observed first order rate constant (kobs) for the reaction of NBD P9 PAI-1 on concentration of scuPA (●), HMW tcuPA (■), Abbokinase (HMW tcuPA) (○), and sctPA (Activase) (▴). Values of kobs were calculated by fitting a double exponential equation to the time traces of changes in NBD-fluorescence emission. The scuPA tracing corresponds to the best fit of a linear equation kobs = 0.0012 s−1. The lines for HMW tcuPA, Abbokinase, and Activase correspond to the best fits (r2 > 0.99) of a hyperbolic equation kobs = klim*[enzyme]/(Km + [enzyme]) to the data shown. The values of klim (the limiting rate of RCL insertion) and Km (concentration of the enzyme at kobs = klim/2) for tcuPA, Abbokinase, and Activase were 23.2 ± 1.1, 20.1 ± 3.3, and 3.1 ± 0.2 s−1, and 3.3 ± 0.3, 4.9 ± 1.0, and 1.9 ± 0.2 μM, respectively.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Concentration Assay, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Rabbit α2M/uPA complexes immunoprecipitated from PFs activate plasminogen and increase uPA activity when exposed to plasmin. A: α2M complexes were isolated from PFs of animals treated with ED scuPA, high dose of Activase, Abbokinase, and with buffer saline vehicle by immunoprecipitation with anti-rabbit α2M IgY (gray) or nonspecific IgY (white). The resin was transferred into 96-well Pro-Bind plates and incubated with plasminogen (0.2 μM) and 0.2 mM chromogenic plasmin substrate (Centerchem, Switzerland) at 37°C. Plasminogen activation was determined as an increase in plasmin (PL) activity in time using SpectraMax Plus (Molecular Devices). The data are presented in the box plot format as described in Fig. 1 legend. Plasminogen activating activity was significantly increased in the PFs of rabbits treated with intrapleural scuPA (P < 0.05). B: effect of plasmin on uPA activity of α2M/uPA complexes precipitated with anti-rabbit α2M IgY from pleural fluids. PFs of animals treated with scuPA were incubated with or without a mixture of 50 nM scuPA and 100 nM PAI-1 in HEPES buffer, pH 7.4, for 5 h at 37°C. α2M/uPA complexes were precipitated with anti-rabbit IgY and Agarose with immobilized anti-IgY goat polyclonal IgG. Amidolytic uPA activity in the resin samples was measured with fluorogenic substrate Pefafluor uPA with (gray) or without (white) preincubation with 1 nM plasmin. *Significant differences (P < 0.05) between the groups.

Article Snippet: Effects of human recombinant suPAR and human plasma α2M on inactivation of uPA by PAI-1. scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Immunoprecipitation, Activity Assay, Isolation, Saline, Incubation, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of uPA with human plasma α2-macroglobulin (α2M), but not with human recombinant suPAR, results in protection from inactivation by PAI-1. scuPA (50 nM) was incubated with 150 nM suPAR, and 50 nM scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) were incubated with 2.0 μM α2M from human plasma in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C for 7 h. At the end of incubation, complexes of uPA with suPAR or α2M were isolated from each reaction mixture by IP with 20 μg of MAb ATN-658 (ATN-658 does not affect amidolytic activity of uPA complexed with suPAR) and with 20 μg of polyclonal sheep anti-human α2M antibodies. Reaction mixtures were incubated with 40 μl of 1:1 slurry of protein A/G Plus Agarose for 2 h at room temperature on a rocker. Reaction mixtures were centrifuged at 3,000–6,000 rpm using an AccuSpin Micro R centrifuge. The resin was washed 3 times with 0.6 ml of cold HEPES buffer, and 50 μl of the resin suspension (total vol 200 μl) was transferred to 96-well plates. Amidolytic uPA activity in precipitated samples was measured with (white bars) or without (gray bars) 80 nM PAI-1. The composition of each reaction mixture, antibodies used for IP, and presence of PAI-1 are shown in the table under the plot.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Clinical Proteomics, Recombinant, Isolation, Activity Assay, Suspension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of scuPA with rabbit PFs forms αM complexes possessing uPA amidolytic activity that resists PAI-1. A: IP of uPA activity by anti-rabbit α2M IgY from PFs of rabbits treated with intrapleural ED of scuPA after preincubation with 50 nM scuPA for 5 h at 37°C in 50 mM HEPES/NaOH, pH 7.4. Samples were immunoprecipitated with α2M IgY or control nonspecific IgY, and uPA amidolytic activity was measured with (white) or without (gray) added exogenous PAI-1 (80 nM) as described in materials and methods and in Fig. 5. uPA amidolytic activity was significantly (*P < 0.05) increased in both groups of PFs from rabbits IP with anti-rabbit α2M vs. those IP nonspecific IgY. B: bar plot of the uPA activity in the precipitates from PFs of animals treated with the ED of scuPA or HD of Activase (Act), Abbokinase (Abb), or vehicle (n = 2 randomly selected samples/each paired group). The precipitation was carried out with 40 μg of either anti-rabbit α2M (filled bars) or nonspecific IgY (hatched bars). The complexes were captured with 80 μl of Agarose slurry and analyzed as described in Fig. 5. C: anti-α2M but not anti-uPAR antibodies precipitate uPA activity from PFs of rabbits, treated with ED of scuPA. IP of uPA activity by polyclonal sheep anti-human α2M antibodies and anti-human uPAR MAb (cross-reacting with rabbit αM and rabbit uPAR, respectively) from PFs of rabbits treated with ED of scuPA. PFs were incubated with 40 μg of antibodies for 2 h in 50 mM HEPES/NaOH, pH 7.4, either with anti-uPAR MAb or with anti-α2M antibodies. In control experiments, PFs were supplemented with exogenous mixtures of 150 nM suPAR with either 50 nM HMW or LMW tcuPA before anti-uPAR MAb was added. Another set of controls contained PFs without addition of antibodies. The composition of each reaction mixture and antibodies used for IP are shown in the table under the plot. Complexes were precipitated with 40 μl of Protein A/G Plus Agarose, and uPA amidolytic activity was measured with (white) or without (gray) 80 nM PAI-1 added, as described in Fig. 5 legend. uPA amidolytic activity was significantly (P < 0.05) increased in PFs incubated with sheep anti-human α2M antibodies and samples supplemented with HMW tcuPA complexed with suPAR (activity without PAI-1) vs. all other samples.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control