Fig 1.
Detection and purification of fibrinolytic component from the aqueous extract of the root of Aristolochia indica.
(A) fibrin zymography. Lane 1, plant extract (35.5 μg); lane 2, plasmin (5 μg). The lanes were selected from two different zymograms run under identical conditions. (B) 2-D fibrin zymography of the root extract (350 μg) showing the presence of multiple components; 5 spots in the acidic zone and one trailing lane in the alkaline zone. (C) Transverse 0–8 M urea gradient fibrin zymography showing presence of multiple enzymes which are stable up to variable denaturant concentrations. (D) dot blot analysis of 1, plasmin; 2, BSA and 3, Russell’s viper venom L-amino acid oxidase; 4, A. indica root extract developed against rabbit polyclonal human plasminogen antibody. (E) DEAE cellulose chromatography of the crude extract where a linear gradient of 0–1 M NaCl was applied. The bar represents the active fractions that were pooled; (F) substrate affinity chromatogram of the pooled fractions from DEAE-chromatography. The bound fractions were eluted by the application of 0-1M NaCl and the bar represents the active fractions. (G) SDS-PAGE of the plant extract (lane 1), unabsorbed fraction from DEAE cellulose column (lane 2), unabsorbed fraction from substrate affinity column (lane 3), absorbed fraction from substrate affinity column (lane 4) and fibrin zymography of the same sample (lane 5). The arrow indicates position of fibrinolysis. The position of the marker proteins is indicated at the left.
Table 1.
Effect of protease inhibitors.
Fig 2.
Difference in fibrinolytic properties of plant enzyme and plasmin.
(A) Time course of degradation of fibrin by the plant enzymes and plasmin as observed from SE-HPLC. 1: Fibrin as control (10 μg); 2–4: fibrin (10 μg) treated with the plant extract (0.1 μg) for 4, 12 and 20 hr. 5–7: fibrin under identical conditions of incubation with plasmin (0.1 μg) for 4, 12 and 20 hr. A Protein Pak 300 SW column equilibrated with 10 mM Na-Phosphate, pH 7.5 containing 100 mM NaCl and at a flow rate of 0.8 ml/min was used. In all sets, the elution was followed at 280 nm. (B-C) time course of degradation of fibrin by the plant enzymes and plasmin as observed in SDS-PAGE. (B) lane 1, plant extract (0.01 μg); lanes 2–8, fibrinogen (10 μg) treated with the plant extract (0.01 μg) for 0, 1, 2, 3, 4, 5 and 6 hr respectively. (C) lane 1, plasmin (0.01 μg); lanes 2–8, fibrinogen (10 μg) treated with plasmin (0.01 μg) for 0, 1, 2, 3, 4, 5 and 6 hr respectively. (D) Time course of amidolysis of S-2251 (0.08 μM) in presence of plasmin (10 μg) (■) and plant enzyme (20 μg) (); (E) Amidolytic assay for plasminogen-like activity of the plant extract (100 μg) in presence plasminogen (60 μg) (
). Plasminogen (60 μg) in presence of urokinase (10 μg) (■) served as control. Spontaneous hydrolysis of S-2251 was insignificant and has not been included to ensure clarity.
Fig 3.
Recovery of thrombosis at rat footpad by the plant fibrinolytic enzymes.
(A-D), after generation of thrombus as described in the text, the following components was applied at 0 hr, (A) PBS (100 μl); (B) plant extract (10 mg/kg); (C) purified enzyme (5 mg/kg) and (D) plasmin (1 mg/kg). In (E), plant extract was applied where no thrombus was generated beforehand to serve as a control to check inherent thrombolytic activity of the extract. Conditions of the corresponding set after 24 h have been illustrated in (F-J). While F shows little recovery out of natural healing, (J) demonstrates that the plant extract is free from thrombolytic activity. Variable degrees of recoveries are evident in (G-I).
Fig 4.
Effect of plant enzyme on morphology of Aβ42 aggregates and fibrin-Aβ42 co-aggregate.
(A) Aβ42 (20 μM) fibrillar form obtained after incubation of Aβ42 peptide (100 μM DMSO solution) in 10 mM sodium phosphate, pH 7.5, containing 100mM NaCl at 37°C with mild shaking for 7 days; (B) fibrin-Aβ42 co-aggregate showing denser and heavily cross-linked structure; (C) preformed Aβ42 aggregate (20 μM) after incubation for 24 hr in presence of the plant enzyme showing presence of intact fibrils; (D) preformed co-aggregate after incubation for 24 hr in presence of the plant enzyme showing complete degradation of the structure leading to monomeric and very small oligomeric forms; (E) preformed co-aggregate incubated for 24 hr in presence of plasmin showing fragmented residual structures that are large, compact and cross-linked. The resolution and magnification 10 nm and 30,000X respectively. (F) fibrin network; (G) Aβ42 oligomer (20 μM) obtained after incubation of Aβ42 peptide (100 μM DMSO solution) in 10 mM sodium phosphate, pH 7.5, containing 100mM NaCl at 37°C with mild shaking for 24 hr (H) Aβ42 aggregate (20 μM); (I) fibrin-Aβ42 co-aggregate; (J) fibrin treated with plant enzyme showing smaller globular fragments; (K) Aβ42 oligomer treated with plant enzyme showing presence of oligomeric forms; (L) Aβ42 aggregate after incubation for 24 hr in presence of the plant enzyme retain its fibriliar structure; (M) the co-aggregate treated with plant enzyme showing random fragmentation of the fibrillar structure, (N) Fibrin treated with plasmin; (O) Aβ42 oligomer treated with plasmin showing smaller monomeric forms; (P) Aβ42 aggregate treated with plasmin for 24 hr showing few fragments along with small monomers; (Q) co-aggregate pretreated with plasmin showing partial trimming of the structures maintaining thick fibrillar network. The horizontal bar shows the amplitude (mV) of the images. The scan size was 1 μm.
Fig 5.
Inhibitory effect of plant enzyme on interaction of fibrin-Aβ42 co-aggregate with neuroblastoma cells
(A) Untreated cells; (B) deposition of preformed fibrin-Aβ42 co-aggregates on cell surface after incubation for 48 hr at 37°C; (C) removal of the co-aggregates from the cell surface after incubation with the plant enzyme; (D) residues of fibrin-Aβ42 co-aggregates on the cell surface after incubation with plasmin. The resolutions of images E-K were variable according to the area of selection to get a clear presentable image. (E) untreated cells with elongated morphology showing prominent blue nuclei (DAPI stained) and very faint expression of Aβ42 appearing as red as Alexa fluor 633 conjugated anti-rabbit secondary antibody was used against rabbit polyclonal antibody to Aβ1–42 peptide; (F) cells treated with fibrin-Aβ42 co-aggregate showing intense greenish-yellow fluorescence (as human fibrinogen conjugated with FITC showing green fluorescence and Aβ42 showing red fluorescence colocalized) indicating penetration of the co-aggregate inside the cells and deposition on the extracellular surface. The cells have markedly different morphology from untreated cells. (G) Cells treated with co-aggregate preincubated with plant enzyme showing morphology and absence of localization of the co-aggregate similar to that of the untreated cells as of (E); (H) cells treated with co-aggregate preincubated with plasmin showing failure of prevention of its localization within the cells and deposition on the cell surface. The morphology also differs from the untreated cells. The magnification of all images was 60X and resolution was 10 nm.
Fig 6.
Reduction of co-aggregate induced cytotoxicity by the plant enzymes.
(A) MTT assay: viability of cells was plotted against the concentration of plant enzyme (black bar) and plasmin (gray bar). The viability of the untreated cells was considered as 100%. (B) LDH assay. The % of LDH released from cells was plotted against the concentration of plant enzyme (black bar) and plasmin (gray bar). LDH released from the co-aggregate treated cells was considered as 100%. The values of the untreated cells were subtracted from the test samples. The bars represent mean ± S.D. of five independent experiments in each set. Probability values of p < 0.05 were considered to represent significant differences. The probability values (p > 0.05) of viability and % LDH release from cells treated with co-aggregate were compared with cells treated with co-aggregates preincubated with plasmin. Insignificant difference between these two groups was observed.
Fig 7.
Topographic AFM images of fibrin-plasma protein co-aggregates.
(A-D) co-aggregates of fibrin-HSA, fibrin-lysozyme, fibrin-transthyretin and fibrin-fibronectin respectively. (E-H) These co-aggregates were treated with plant enzyme for 24 hr and their corresponding morphological features were shown. (I-L) The morphology of the co-aggregates treated with plasmin for 24 hr are illustrated. Features have been described in the text. The instrumentation was same as described in Fig 5.
Fig 8.
Hypothetical model of fibrin-Aβ42 co-aggregate formation and its destabilization by the plant enzymes (PE) or plasmin (PL).
Fibrinogen and Aβ42 form aggregates independently that are degraded by PL. PE also degrades fibrin clot. When fibrinogen interacts with Aβ42, it forms abnormal co-aggregate. The efficiency of PE is superior to PL in destabilizing the co-aggregate. FDP stands for fibrin degradation products. Stable end products are marked bold.