Journal: International Journal of Molecular Sciences

Article Title: Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

doi: 10.3390/ijms24010327

Figure Lengend Snippet: Temporal changes in LDL-EV hemostatic protein composition in post-AMI patients with adverse and reverse LV remodeling. Coagulation proteins (VWF, SerpinC1) and fibrinolytic protein (plasminogen) levels and their ratios (VWF:Plasminogen, SerpinC1:Plasmingen) in LDL-EVs of 198 post-AMI patients at baseline and after 1 and 6 month follow-up. ( A ) A diagram illustrating the studied hemostatic proteins in LDL-EVs. ( B – F ) Differences between baseline and follow-up measurements were established by Wilcoxon signed-ranked test (horizontal statistical bar). Differences in the three protein levels and the protein ratios between patients with adverse LV remodeling and reverse LV remodeling were established by Mann–Whitney U test (vertical statistical bar). Data are presented as mean ± SEM.

Article Snippet: The antibodies and recombinant proteins were as follows: for detection of VWF we used recombinant human VWF protein (Factor VIII free, Fitzgerald #30C-CP4003U, Fitzgerald Industries International, Acton, MA, USA), anti-human VWF (Fitzgerald #70R-10589, Fitzgerald Industries International, Acton, MA, USA), and biotinylated anti-human VWF (Fitzgerald #60R-1019, Fitzgerald Industries International, Acton, MA, USA); for detection of SerpinC1, anti-thrombin III antibody (NOVUS Biologicals #NBP1-05149, Littleton, CO, USA), human SerpinC1 biotinylated affinity purified antibody (R&D Systems #BAF1267, Minneapolis, MN, USA) and recombinant human SerpinC1 (R&D Systems #1267-PI-010, Minneapolis, MN, USA); for detection of plasminogen, anti-human plasminogen (NOVUS Biologicals NB120-10176, Littleton, CO, USA), biotinylated anti-human plasminogen (NOVUS Biologicals, NB120-10177, Littleton, CO, USA), and recombinant human plasminogen (R&D system,1939-SE-200, Minneapolis, MN, USA); and for detection of SerpinF2, anti-human SerpinF2 (R&D Systems #MAB1470, Minneapolis, MN, USA), biotinylated anti-human SerpinF2 (R&D Systems #BAF1470, Minneapolis, MN, USA) and recombinant human SerpinF2 (R&D Systems #1470-PI-010, Minneapolis, MN, USA).

Techniques: Coagulation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

doi: 10.3390/ijms24010327

Figure Lengend Snippet: ML-SEM modeling for LDL-EV proteins.

Article Snippet: The antibodies and recombinant proteins were as follows: for detection of VWF we used recombinant human VWF protein (Factor VIII free, Fitzgerald #30C-CP4003U, Fitzgerald Industries International, Acton, MA, USA), anti-human VWF (Fitzgerald #70R-10589, Fitzgerald Industries International, Acton, MA, USA), and biotinylated anti-human VWF (Fitzgerald #60R-1019, Fitzgerald Industries International, Acton, MA, USA); for detection of SerpinC1, anti-thrombin III antibody (NOVUS Biologicals #NBP1-05149, Littleton, CO, USA), human SerpinC1 biotinylated affinity purified antibody (R&D Systems #BAF1267, Minneapolis, MN, USA) and recombinant human SerpinC1 (R&D Systems #1267-PI-010, Minneapolis, MN, USA); for detection of plasminogen, anti-human plasminogen (NOVUS Biologicals NB120-10176, Littleton, CO, USA), biotinylated anti-human plasminogen (NOVUS Biologicals, NB120-10177, Littleton, CO, USA), and recombinant human plasminogen (R&D system,1939-SE-200, Minneapolis, MN, USA); and for detection of SerpinF2, anti-human SerpinF2 (R&D Systems #MAB1470, Minneapolis, MN, USA), biotinylated anti-human SerpinF2 (R&D Systems #BAF1470, Minneapolis, MN, USA) and recombinant human SerpinF2 (R&D Systems #1470-PI-010, Minneapolis, MN, USA).

Techniques: Coagulation

Journal: International Journal of Molecular Sciences

Article Title: Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction

doi: 10.3390/ijms24010327

Figure Lengend Snippet: Area under the receiver-operating curve (AUC) of different candidate markers predicting reverse LV remodeling.

Article Snippet: The antibodies and recombinant proteins were as follows: for detection of VWF we used recombinant human VWF protein (Factor VIII free, Fitzgerald #30C-CP4003U, Fitzgerald Industries International, Acton, MA, USA), anti-human VWF (Fitzgerald #70R-10589, Fitzgerald Industries International, Acton, MA, USA), and biotinylated anti-human VWF (Fitzgerald #60R-1019, Fitzgerald Industries International, Acton, MA, USA); for detection of SerpinC1, anti-thrombin III antibody (NOVUS Biologicals #NBP1-05149, Littleton, CO, USA), human SerpinC1 biotinylated affinity purified antibody (R&D Systems #BAF1267, Minneapolis, MN, USA) and recombinant human SerpinC1 (R&D Systems #1267-PI-010, Minneapolis, MN, USA); for detection of plasminogen, anti-human plasminogen (NOVUS Biologicals NB120-10176, Littleton, CO, USA), biotinylated anti-human plasminogen (NOVUS Biologicals, NB120-10177, Littleton, CO, USA), and recombinant human plasminogen (R&D system,1939-SE-200, Minneapolis, MN, USA); and for detection of SerpinF2, anti-human SerpinF2 (R&D Systems #MAB1470, Minneapolis, MN, USA), biotinylated anti-human SerpinF2 (R&D Systems #BAF1470, Minneapolis, MN, USA) and recombinant human SerpinF2 (R&D Systems #1470-PI-010, Minneapolis, MN, USA).

Techniques:

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).

Article Snippet: Recombinant SERPINE1 protein (PAI-1, 1786-PI), SERPINA1 protein (A1AT, 1268-PI), SERPINC1 protein (ATIII, 1267-PI), and nafamostat mesylate (3081) were purchased from R&D Systems.

Techniques: Infection, Inhibition, Transfection, Expressing, Synthesized, SPR Assay, Binding Assay, Positive Control, Protease Inhibitor, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

doi: 10.3390/ijms19010094

Figure Lengend Snippet: Coagulation proteins enriched in the very-low- and low-density lipoprotein ((V)LDL) and high-density lipoprotein (HDL) precipitates. von Willebrand factor (VWF), SerpinC1, plasminogen (PLG) and SerpinF2 levels in the (V)LDL and HDL precipitates were determined by multiplex immunoassay and normalized to the original volumes of plasma. n = 20, *** p < 0.001 compared to the respective protein levels in the HDL precipitate. Bars represent mean ± standard error of mean (S.E.M).

Article Snippet: Detection of von Willebrand factor (VWF) used recombinant human VWF protein (Factor VIII free, Fitzgerald #30C-CP4003U, Fitzgerald Industries International, Acton, MA, USA), anti-human VWF (Fitzgerald #70R-10589, Fitzgerald Industries International, Acton, MA, USA), and biotinylated anti-human VWF (Fitzgerald #60R-1019, Fitzgerald Industries International, Acton, MA, USA); detection of SerpinC1 used antithrombin III antibody (NOVUS Biologicals #NBP1-05149, Littleton, CO, USA), human SerpinC1 biotinylated affinity purified antibody (R&D Systems #BAF1267, Minneapolis, MN, USA), and recombinant human SerpinC1 (R&D Systems #1267-PI-010, Minneapolis, MN, USA); detection of plasminogen used anti-human plasminogen (HyTest #4P11, Turku, Finland), biotinylated anti-human plasminogen (HyTest #4P11B, Turku, Finland), and recombinant human plasminogen (BBI Solutions #P204-1; Cardiff, UK); and detection of SerpinF2 used anti-human SerpinF2 (R&D Systems #MAB1470, Minneapolis, MN, USA), biotinylated anti-human SerpinF2 (R&D Systems #BAF1470, Minneapolis, MN, USA), and recombinant human SerpinF2 (R&D Systems #1470-PI-010, Minneapolis, MN, USA).

Techniques: Coagulation, Multiplex Assay, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

doi: 10.3390/ijms19010094

Figure Lengend Snippet: Distribution of VWF, SerpinC1, PLG, SerpinF2, and ApoB in density sub-fractions of the (V)LDL precipitate after density gradient centrifugation. * V1, the first sub-fraction of (V)LDL precipitate. The density of each sub-fraction was indicated accordingly. ** The unit of density is g/mL. ( A ) Protein levels of VWF, SerpinC1, PLG, and SerpinF2 are presented as the percentage of the 8th sub-fraction (V8). Protein levels of ApoB are presented as the percentage of the 4th sub-fraction (V4). ( B ) A representative electron microscope (EM) image of the 4th sub-fraction shows lipoprotein particles. Scale bar = 500 nm. ( B’ ) Larger magnification of insert in ( B ). Scale bar = 50 nm. ( C ) A representative EM image of the 7th sub-fraction shows EVs. Scale bar = 100 nm.

Article Snippet: Detection of von Willebrand factor (VWF) used recombinant human VWF protein (Factor VIII free, Fitzgerald #30C-CP4003U, Fitzgerald Industries International, Acton, MA, USA), anti-human VWF (Fitzgerald #70R-10589, Fitzgerald Industries International, Acton, MA, USA), and biotinylated anti-human VWF (Fitzgerald #60R-1019, Fitzgerald Industries International, Acton, MA, USA); detection of SerpinC1 used antithrombin III antibody (NOVUS Biologicals #NBP1-05149, Littleton, CO, USA), human SerpinC1 biotinylated affinity purified antibody (R&D Systems #BAF1267, Minneapolis, MN, USA), and recombinant human SerpinC1 (R&D Systems #1267-PI-010, Minneapolis, MN, USA); detection of plasminogen used anti-human plasminogen (HyTest #4P11, Turku, Finland), biotinylated anti-human plasminogen (HyTest #4P11B, Turku, Finland), and recombinant human plasminogen (BBI Solutions #P204-1; Cardiff, UK); and detection of SerpinF2 used anti-human SerpinF2 (R&D Systems #MAB1470, Minneapolis, MN, USA), biotinylated anti-human SerpinF2 (R&D Systems #BAF1470, Minneapolis, MN, USA), and recombinant human SerpinF2 (R&D Systems #1470-PI-010, Minneapolis, MN, USA).

Techniques: Gradient Centrifugation, Microscopy

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Identification and function probing of an antithrombin IIIβ conformation-specific antibody.

doi: 10.1111/jth.13198

Figure Lengend Snippet: Fig. 4. The binding of TPP2009 to EA-hy926 endothelial cells is dependent on antithrombin III (AT)b and glycosaminoglycan (GAG). (A) Dose-dependent binding of TPP2009 on EA-hy926 cells after addition of exogenous ATb. The y-axis represents the fold increase in median flu- orescence intensity (MFI) relative to cells not pretreated with AT. (B) No binding of an isotype IgG control on EA-hy926 cells. (C) GAG removal (digestion for 2 h) reduced the binding of TPP2009 solely to EA-hy926 cells pretreated with ATb. (D) The binding of AF1267, an antibody against both ATa and ATb, to EA-hy926 cells pretreated with ATa or ATb was reduced after GAG removal. (E) An IgG isotype control showed minimal binding to AT-pretreated cells, and its binding was not affected by GAG removal.

Article Snippet: Human AT-depleted (hAT-DP) and FVIIIdeficient human plasma were from Haematologic Technologies (Essex Junction, VT, USA), and AF1267 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Control

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Protective role of protein C inhibitor in monocrotaline-induced pulmonary hypertension.

doi: 10.1111/j.1538-7836.2006.02174.x

Figure Lengend Snippet: Fig. 5. Markers of coagulation and fibrinolysis system. Markers of coagulation and fibrinolysis systems were measured in bronchoalveolar lavage fluid using commercial enzyme immunoassay kits in wild-type mice treated with saline (WT/SAL, n ¼ 15) or monocrotaline (WT/MCT, n ¼ 13) and in protein C inhibitor transgenic mice treated with saline (TG/SAL, n ¼ 8) or monocrotaline (TG/MCT, n ¼ 11). (A) The level of thrombin– antithrombin complex was significantly different between WT/SAL and WT/MCT, between TG/SAL and TG/MCT, and between WT/MCT and TG/MCT mice. (B) The level of plasminogen activator inhibitor-1 was significantly different between WT/SAL and WT/MCT mice, and between TG/SAL and TG/MCT mice, but not between WT/MCT and TG/MCT mice or between WT/SAL and TG/SAL mice. (C) The level of tissue-type plasminogen activator was significantly different between WT/MCT and TG/MCT mice but not between WT/SAL and WT/MCT, TG/SAL and TG/MCT or WT/SAL and TG/SAL mice. Samples were tested in dupli- cate. Bars represent the means ± SEM. Statistical analysis was performed by ANOVA with Fisher’s predicted least significant difference test.

Article Snippet: The concentration of PCI-thrombin complex was measured by EIA using antithrombin antibody (Acris antibodies GmbH, Hiddenhausen, Germany) and biotin-labeled anti-PCI antibody.

Techniques: Coagulation, Enzyme-linked Immunosorbent Assay, Saline, Transgenic Assay