Journal: International Journal of Molecular Sciences

Article Title: Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin

doi: 10.3390/ijms24119306

Figure Lengend Snippet: Catalytically active testisin accelerates prothrombin-dependent fibrin generation through a FXa-dependent mechanism. ( a ) Testisin-induced fibrin generation is dependent upon its catalytic activity. TsWT, TsMut, and Ctl cells were treated with 1 mg/mL fibrinogen with 10 nM prothrombin zymogen and turbidity at OD 350 monitored over 5 h. Graph shows average OD 350 from quadruplicate wells ± SEM and is representative of at least 5 independent experiments. ( b ) SDS-PAGE of urea-solubilized lysates confirms faster generation of fibrin in TsWT cells compared to Ctl and TsMut cells. Lysates taken at 0.5, 1, and 3 h from the experiment ( a ) stained with Coomassie blue are shown. Shown also are γ-γ dimers (black arrows), monomer fibrinogen γ-chain, α-polymers (white arrows), and the thrombin-cleaved β-chain (*). Control lanes show cell-free fibrinogen alone (Fg, lane 1), fibrinogen and prothrombin (Fg + PT, lane 2), and polymerized fibrin (Fg + Thr, lane 3). ( c ) TsWT cells stimulate prothrombin activation. Thrombin activity was measured using the Bz-FVR-AMC fluorogenic substrate on cells treated with fibrinogen alone (−PT) or with 10 nM prothrombin (+PT) and fluorescence measured at Ex370 nm/Em450 nm. Graph shows average thrombin activity normalized to Ctl + Fg alone ± SEM at 3 h from 4–6 independent experiments. ( d ) Compared to thrombin, testisin catalyzes only a low level of proteolytic cleavage of the fluorogenic thrombin substrate Bz-FVR-AMC. Activity (relative fluorescence units, RFU) of α-thrombin (1 nM), rTs (10 nM) or rTs control (Cont., (10 nM) see Materials and Methods) was measured from duplicate wells over 20 min. Graph is representative of 4 independent experiments. ( e ) Average turbidity measurements at 3 h from 5–6 independent experiments in Ctl, TsWT, and TsMut cells in the presence of fibrinogen, with (+) or without (−) 10 nM prothrombin (PT). ( f ) Rivaroxaban does not inhibit cell-expressed testisin. Testisin activity was assayed in the presence of 1 mg/mL fibrinogen with (+) or without (−) 50 nM rivaroxaban using the Boc-QAR-AMC substrate. Graph shows average testisin activity at 3 h ± SEM from 3 independent experiments. Data are normalized to Ctl + Fg alone. ( g ) Rivaroxaban dose-dependently inhibits prothrombin-dependent induction of turbidity mediated by TsWT cells. Turbidity assay in the presence of fibrinogen and prothrombin in which TsWT cells were treated with 20 or 50 nM rivaroxaban or no inhibitor. Turbidity was monitored over 3 h. Graph represents mean ± SEM from triplicate wells. ( h ) Average turbidity induced by Ctl, TsWT and TsMut cells in the absence (−) or presence (+) of 50 nM rivaroxaban (RIV) at 3 h ± SEM from 3–4 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; ns, non-significant.

Article Snippet: Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [ ]), uPA (Glt-GR-AMC [ ]), plasmin (Boc-EEK-AMC [ ]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland.

Techniques: Activity Assay, SDS Page, Staining, Activation Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin

doi: 10.3390/ijms24119306

Figure Lengend Snippet: Acceleration of fibrinolysis by GPI-anchored testisin is dependent upon plasmin (ogen). ( a ) Time course analysis of cell-based fibrinolysis assays. TsWT, TsMut, and Ctl cells were treated with fibrinogen, without (closed symbols) or with 250 µM TXA (open symbols) and fibrin polymerization initiated with thrombin. Turbidity was monitored over 24 h. Data are representative of 3 independent experiments and show average turbidity ± SEM of quadruplicate wells. ( b ) TXA does not inhibit the activity of cell-expressed testisin, assessed in the presence of 1 mg/mL fibrinogen with (+) or without (−) 250 µM TXA using the peptide substrate Boc-QAR-AMC. Graph shows average activity ± SEM from 5 independent experiments at 3 h after adding substrate and is normalized to Ctl -TXA. ( c ) Average turbidity at the 24 h endpoint in the absence (−) or presence (+) of 250 µM TXA from 3 independent experiments. Data are expressed as % maximal turbidity reached after addition of thrombin in each experimental treatment. ( d ) SDS-PAGE confirms faster fibrin degradation by TsWT cells compared to Ctl and TsMut. Urea-solubilized lysates prepared at the 24 h endpoint analyzed by reducing SDS-PAGE stained with Coomassie blue. Control lanes include fibrin alone (Fn, lane 1), fibrin generated in the presence of 20 nM plasminogen (Fn + Plg, lane 2), and fibrin generated in the presence of plasminogen to which 5 nM uPA was added after polymerization to generate plasmin (Fn + Plg + uPA, lane 3). Fibrin degradation is shown by loss of insoluble fibrin (gray triangle) and α-polymers (white triangles). γ-γ dimers (black triangle) and the β-chain are also cleaved, producing γ-γ deg and β-deg fibrin degradation products, respectively (lane 3). Fibrin degradation species were identified as reported in . Graphs show quantitation of fibrin degradation products by densitometry, expressed as the percentage of the total protein quantified in each sample. ( e ) Cellular testisin facilitates plasmin(ogen)-dependent cell invasion through fibrin. TsWT, TsMut, and Ctl cells were plated onto fibrin or Matrigel-coated Transwells in serum-free medium with (+) or without (−) 250 µM TXA, and invasion towards 10% serum-containing media assessed. Representative images shown are stitched, whole-well scans of the underside of Transwells showing invaded cells stained with KwikDiff after 10 h invasion (4× original magnification). The two left panels (−TXA) show wells from 2 independent experiments; right panel (+TXA) shows wells from a representative experiment showing inhibition of testisin-mediated fibrin invasion. Images are representative of 3 independent experiments for both conditions. ( f ) Quantitation of cell invasion analyzed by manual cell counting of all invaded cells/membrane using Image J. TsWT invaded through fibrin ~13-fold faster than Ctl and TsMut cells, but invasion through Matrigel was not enhanced by expression of wildtype testisin. Data show average fold invasion relative to Ctl from 2–3 independent experiments, * p < 0.05; *** p < 0.005; **** p < 0.001; ns, non-significant.

Article Snippet: Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [ ]), uPA (Glt-GR-AMC [ ]), plasmin (Boc-EEK-AMC [ ]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland.

Techniques: Activity Assay, SDS Page, Staining, Generated, Quantitation Assay, Inhibition, Cell Counting, Expressing