α 2 antiplasmin  (Assaypro)

Journal: Frontiers in Immunology

Article Title: The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction

doi: 10.3389/fimmu.2024.1445294

Figure Lengend Snippet: The 4G allele of the PAI-1 promoter is associated with higher PAI-1 transcript and lower plasmin activity in COVID-19 patients. (A) PAI-1 levels as measured by ELISA in the study cohort (n=43). (B) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of all patient samples showed allele-specific fragments after restriction enzyme treatment. (C) Examples of Sanger Sequencing for confirmation of the identified PAI-1 4G/5G rs1799889 and +43G>A (rs6092) polymorphisms. In the study group, we did not find the 43AA genotype. Therefore, no data were available (not found, N/F) for this genotype in panels (D–G) . (D) Fold change in PAI-1 expression in peripheral mononuclear cells (PBMCs) of COVID-19 patients sorted by genotype (n=42). The expression of indicated genes is normalized to the endogenous reference β-actin and presented as a relative fold change to the expression in 4G/4G groups according to the comparative Ct method (2−ΔΔCt). Experiments were repeated twice with samples run in triplicates each. (E) Circulating total PAI-1 protein sorted according to genotypes (data set is the same as in panel (A) . (F) PAI-1 activity after sorting in genotypes as determined by ELISA [reanalysis of original data published ]. (G) Serum plasmin/a2-antiplasmin complex levels by ELISA. Dots in each panel represent data from one patient sample. N.F., not found, N/A, not available. Data represent mean ± SEM with p values from unpaired Student’s t-test or the Mann-Whitney test. # p<0.05; * p<0.05; ** p<0.01; *** <0.005; n/s, not significant.

Article Snippet: Then membranes were probed with one of the following primary Abs: uPAR rabbit polyclonal Abs (1 ug/ml ( ); Plg rabbit polyclonal Abs (0,5 ug/ml, ( ); α2-antiplasmin rabbit polyclonal antibodies (1:1000, #sc73659, Santa Cruz, Dallas, USA).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Sequencing, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction

doi: 10.3389/fimmu.2024.1445294

Figure Lengend Snippet: IL-1β establishes an anti-fibrinolytic gene expression profile in 4G4G primary endothelial cells (ECs). (A) Transcriptional activation of the human PAI-1 promoter by recIL-1β. HUVEC cells were transiently transfected with the PAI-1 4G and 5G luciferase reporter plasmids. Cells were treated with or without IL-1β for 24 hours before lysis. Firefly luciferase activity was normalized to Renilla luciferase activity and is expressed as fold change to controls. Data shown are the mean ± SEM of triplicates from a representative experiment (n=3/group). (B–G) Fold change in PAI-1 (B) , tPA (C) , and uPA (D) , KLF2 (E) , NFκB (F) , and TGFb (G) expression in cultured 4G4G, 4G5G, or 5G5G ECs stimulated with rec IL-1β (two independent experiments using two different cell origins per genotype were performed; data shown are from one experiment; n=4/group). The expression of the indicated genes is normalized to the endogenous reference β-actin and presented as a relative fold change to expression in the control expression of each genotype according to the comparative Ct method (2−ΔΔCt). (H) Immunoblot of plasmin/α2-antiplasmin complex (PAP) in an equal volume of supernatants of EC cultures. (I) Band intensity quantified of the G blot, whereby each recIL-1β sample was normalized to its control samples. (J–L) A representative immunoblot of Plg using supernatants from cultures treated with or without (control) IL-1β showed angiostatin fragments (J) after loading an equal volume of supernatants from EC cultures. Band intensity quantified of the I blot, whereby each recIL-1β sample was normalized to its control samples, showing the Plg cleavage fragment angiostatin at 38 kDa (K) and 50 kDa (L) . All western blots were performed at least twice with similar results. # p<0.05; ## p<0.01; * p<0.05; ** p<0.01; *** p<0.005; n/s, not significant. In vitro data were presented as box plots to discriminate in vivo data from the following in vitro data. All experiments were done in triplicate, and two cell lines for each genotype were used.

Article Snippet: Then membranes were probed with one of the following primary Abs: uPAR rabbit polyclonal Abs (1 ug/ml ( ); Plg rabbit polyclonal Abs (0,5 ug/ml, ( ); α2-antiplasmin rabbit polyclonal antibodies (1:1000, #sc73659, Santa Cruz, Dallas, USA).

Techniques: Gene Expression, Activation Assay, Transfection, Luciferase, Lysis, Activity Assay, Expressing, Cell Culture, Control, Western Blot, In Vitro, In Vivo

Journal: Cells

Article Title: PAI-1 Overexpression in Valvular Interstitial Cells Contributes to Hypofibrinolysis in Aortic Stenosis

doi: 10.3390/cells12101402

Figure Lengend Snippet: Valvular expression of fibrinolytic proteins and inhibitors. Representative microphotographs showing valvular expression of plasminogen, α 2 -antiplasmin, tissue plasminogen activator (tPA), and D-dimer in control (left panel) and stenotic aortic leaflets (right panel). Red arrowhead indicates aortic side of the leaflet; yellow arrowheads indicate the immunopositive areas. Scale bar 200 μm, original magnification 4×.

Article Snippet: Immunofluorescence was conducted according to the previously described protocol [ ] using primary antibodies against PAI-1 (1:500; Abcam, Cambridge, UK), tPA (1:100; Novus Biologicals, Centennial, CO, USA), α 2 -antiplasmin (1:250; Santa Cruz Biotechnology, Dallas, TX, USA), plasminogen (1:500; GeneTex, Irvine, CA, USA), fibrin degradation products (D-dimer, 1:100; Bioss Antibodies, Woburn, MA, USA) and NF-κB (p65, 1:500, Abcam) and the corresponding secondary donkey or goat antibodies conjugated with AlexaFluor 488 or 594 (Abcam) (1:1000).

Techniques: Expressing, Control

Journal: Cells

Article Title: PAI-1 Overexpression in Valvular Interstitial Cells Contributes to Hypofibrinolysis in Aortic Stenosis

doi: 10.3390/cells12101402

Figure Lengend Snippet: Expression of fibrinolytic proteins in valve interstitial cells (VICs) according to different culture conditions. Representative microphotographs of VICs cultured in different conditions tested for ( A ) PAI-1 expression and IgG isotype control, ( B ) plasminogen, α 2 -antiplasmin or tPA expression, and IgG isotype control. Cell nuclei are stained blue (DAPI), protein expression is green. Scale bar 20 µm, original magnification 40×. The experiment was repeated three times using VICs isolated from different valves.

Article Snippet: Immunofluorescence was conducted according to the previously described protocol [ ] using primary antibodies against PAI-1 (1:500; Abcam, Cambridge, UK), tPA (1:100; Novus Biologicals, Centennial, CO, USA), α 2 -antiplasmin (1:250; Santa Cruz Biotechnology, Dallas, TX, USA), plasminogen (1:500; GeneTex, Irvine, CA, USA), fibrin degradation products (D-dimer, 1:100; Bioss Antibodies, Woburn, MA, USA) and NF-κB (p65, 1:500, Abcam) and the corresponding secondary donkey or goat antibodies conjugated with AlexaFluor 488 or 594 (Abcam) (1:1000).

Techniques: Expressing, Cell Culture, Control, Staining, Isolation