Journal: PLoS Pathogens

Article Title: Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

doi: 10.1371/journal.ppat.1003470

Figure Lengend Snippet: Kallikrein-kinin system and BK inhibitors. A schematic illustration of three inhibitors that impact BK-mediated increase in endothelial cell permeability. CTI (corn trypsin inhibitor) inhibits FXIIa; PKSI-527 (plasma kallikrein specific inhibitor) inhibits KAL activity; Icatibant (HOE 140) is a peptidomimetic drug consisting of ten amino acids, which is a selective and specific antagonist of BKB2 receptors and is used in humans under orphan drug status for symptomatic treatment of hereditary angioedema.

Article Snippet: PKSI-527 is a specific inhibitor of KAL activity (Enzo Life Sciences) and corn trypsin inhibitor (CTI) inhibits FXIIa (Haematologic Technologies Inc.).

Techniques: Permeability, Activity Assay

Journal: PLoS Pathogens

Article Title: Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

doi: 10.1371/journal.ppat.1003470

Figure Lengend Snippet: Kallikrein-kinin system activation and changes in permeability of hantavirus-infected HUVEC. Mock-, HTNV-, or ANDV-infected HUVEC were trypsinized, seeded onto ECIS chamberslides, and cultured until confluent. Media were removed and replaced with phenol red free EBM containing Zn 2+ . After an equilibration, 0.2 ml of phenol red free EBM containing Zn 2+ , FXII, PK, and HK (100 nM each) were added to cells (time zero) (black lines) (A). To measure inhibition of activation, some samples were treated with CTI (1 µM) (blue line), PKSI-527 (5 µM) (red line), or HOE 140 (1 µM) (green line) simultaneously with factors (B, C, D). Real time measurements frequency measurements were taken throughout the assay.

Article Snippet: PKSI-527 is a specific inhibitor of KAL activity (Enzo Life Sciences) and corn trypsin inhibitor (CTI) inhibits FXIIa (Haematologic Technologies Inc.).

Techniques: Activation Assay, Permeability, Infection, Electric Cell-substrate Impedance Sensing, Cell Culture, Inhibition

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial plasminogen activator inhibitor-1 blocks the intrinsic pathway of coagulation, inducing the clearance and degradation of factor XIa.

doi: 10.1161/ATVBAHA.119.312619

Figure Lengend Snippet: Detection of FXIa or kallikrein generation by FXIIa on the surface of HUVECs. ( A ) HK (100 nM) and FXI (100 nM) or ( B ) PK (100 nM) and HK (100 nM) were incubated with FXIIa (1 nM) for 1 hr in the presence ( middle panel ) or absence ( left panel ) of HUVECs. Reactions were stopped with CTI (40 μg/ml). FXIa and kallikrein activity was determined by adding S-2366 (0.8 mM) or S-2302 (0.6 mM), respectively; the rate of substrate hydrolysis was measured at 405 nm. Empty wells or HUVECs were incubated with increasing concentrations of ( A, right panel ) FXIa or ( B, right panel ) kallikrein for 2 hrs at 37°C FXIa and kallikrein activity was determined by adding S-2366 (0.8 mM) or S2302 (0.6 mM), respectively and the rate of substrate hydrolysis was measured at 405 nm. ( C ) HUVECs were incubated for 16 hrs in the absence (□) or presence of TNFα (0.5 ng/ml) (○) or incubated for 4 hrs with elastase (50 nM) (△). Subsequently, cells were incubated with increasing concentrations of FXIa. FXIa activity was determined by adding S-2366 (0.6 mM) and the rate of substrate hydrolysis was measured at 405 nm. ( D ) HUVECs were incubated for 3 hrs in the absence or presence of heparinase I, II and III (1 U/ml) followed by cell surface detection of heparan sulfate using an anti-10E4 epitope antibody. ( E ) HUVECs were incubated for 3 hrs in the absence (□) or presence (○) of heparinase I, II and III (1 U/ml). Subsequently, cells were incubated with increasing concentrations of FXIa. FXIa activity was determined by adding S-2366. ( F ) HUVECs were incubated for 4 hrs in the absence (□) or presence of thrombin (20 nM) (○). Subsequently, cells were incubated with increasing concentrations of FXIa. FXIa activity was determined by adding S-2366. Data are mean ± SD (n = 3). * Indicates between-groups differences with P

Article Snippet: Plasma-derived FXIIa, PK, kallikrein, HK and corn trypsin inhibitor (CTI) were from Enzyme Research Laboratories, Inc. (South Bend, IN, USA).

Techniques: Incubation, Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial plasminogen activator inhibitor-1 blocks the intrinsic pathway of coagulation, inducing the clearance and degradation of factor XIa.

doi: 10.1161/ATVBAHA.119.312619

Figure Lengend Snippet: Detection of FXIa activity in the presence of a blocking anti-PAI-1 antibody. ( A ) Empty wells (●, ○) or HUVECs (■, □) were incubated with FXIa in the absence (□, ○) or presence (■, ●) of a blocking anti-PAI-1 antibody (20 μg/ml) for 2 hrs. at 37°C. FXIa activity was determined by adding S-2366. ( B ) HUVECs were incubated for 16 hrs with TNFα (0.5 ng/ml). Subsequently, cells were incubated with FXIa (500 pM) in the absence (black bars) or presence (white bars) of an anti-PAI-1 antibody (20 μg/ml) for 2 hrs at 37°C. FXIa activity was determined by adding S-2366. ( C ) Empty wells ( ○ ) or HUVECs (□, ●) were incubated with FXIa in the absence (□) or presence (●) of t-PA (30 nM) for 2 hrs at 37°C. FXIa activity was determined by adding S-2366 ( D ) Increasing concentrations of rPAI-1 were incubated with 5 nM FXIa, kallikrein or t-PA at 37°C for 30 min and activity was determined by adding S-2366, S2302 or S2288 respectively. ( E ) PAI-1 detection by western blot in ECs or platelets supernatant. ( F ) FXIa (150 pM) were incubated with vehicle or supernatant from activated platelets in the presence (white bars) or absence (black bars) of an anti-PAI-1 antibody. ( G ) HUVECs were incubated with 0.1 nM FXIa in the presence or absence of a blocking anti-PAI-1 antibody (20 μg/ml) for 2 hrs at 37°C. Fibrin generation was determined in recalcified PPP in the presence of CTI and a blocking anti-TF antibody. ( H ) HUVECs were incubated with TNFα. Fibrin generation was determine in PPP in the presence of CTI or FXI depleted plasma (FXI-dep) in the presence or absence of an anti-TF, an anti-FXI or an anti-PAI-1 antibody. Data are mean ± SD (n = 3). *P

Article Snippet: Plasma-derived FXIIa, PK, kallikrein, HK and corn trypsin inhibitor (CTI) were from Enzyme Research Laboratories, Inc. (South Bend, IN, USA).

Techniques: Activity Assay, Blocking Assay, Incubation, Western Blot