Hypothetical model of fibrin-Aβ42 co-aggregate formation and its destabilization by the plant enzymes (PE) or plasmin (PL).

<p>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.</p

Inhibitory effect of plant enzyme on interaction of fibrin-Aβ42 co-aggregate with neuroblastoma cells

<p><b>(A)</b> Untreated cells; <b>(B)</b> deposition of preformed fibrin-Aβ42 co-aggregates on cell surface after incubation for 48 hr at 37°C; <b>(C)</b> removal of the co-aggregates from the cell surface after incubation with the plant enzyme; <b>(D)</b> 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. <b>(E)</b> 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; <b>(F)</b> 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. <b>(G)</b> 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 <b>(E)</b>; <b>(H)</b> 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.</p

Detection and purification of fibrinolytic component from the aqueous extract of the root of <i>Aristolochia indica</i>.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> Transverse 0–8 M urea gradient fibrin zymography showing presence of multiple enzymes which are stable up to variable denaturant concentrations. <b>(D)</b> dot blot analysis of 1, plasmin; 2, BSA and 3, Russell’s viper venom L-amino acid oxidase; 4, <i>A</i>. <i>indica</i> root extract developed against rabbit polyclonal human plasminogen antibody. <b>(E)</b> 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; <b>(F)</b> 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. <b>(G)</b> 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.</p

An Enzyme from <i>Aristolochia indica</i> Destabilizes Fibrin-β Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases

<div><p>Fibrinogen and β-amyloid (Aβ) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant <i>Aristolochia indica</i> that showed fibrinolytic as well as fibrin-Aβ co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aβ, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme.</p></div

Recovery of thrombosis at rat footpad by the plant fibrinolytic enzymes.

<p><b>(A-D)</b>, after generation of thrombus as described in the text, the following components was applied at 0 hr, <b>(A)</b> PBS (100 μl); <b>(B)</b> plant extract (10 mg/kg); <b>(C)</b> purified enzyme (5 mg/kg) and <b>(D)</b> plasmin (1 mg/kg). In <b>(E)</b>, 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 <b>(F-J)</b>. While F shows little recovery out of natural healing, <b>(J)</b> demonstrates that the plant extract is free from thrombolytic activity. Variable degrees of recoveries are evident in <b>(G-I)</b>.</p

Topographic AFM images of fibrin-plasma protein co-aggregates.

<p><b>(A-D)</b> co-aggregates of fibrin-HSA, fibrin-lysozyme, fibrin-transthyretin and fibrin-fibronectin respectively. <b>(E-H)</b> These co-aggregates were treated with plant enzyme for 24 hr and their corresponding morphological features were shown. <b>(I-L)</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141986#pone.0141986.g005" target="_blank">Fig 5</a>.</p

Effect of protease inhibitors.

<p>^<b>a</b> The residual activity of the inhibitor treated sample was calculated with respect to the activity of enzyme in absence of any inhibitor which was considered as 100%. The concentrations of protease inhibitors were 1 mM for all the sets. The values represent mean ± S.D., where <i>p</i> < 0.05.</p><p>Effect of protease inhibitors.</p

Reduction of co-aggregate induced cytotoxicity by the plant enzymes.

<p><b>(A)</b> 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>(B)</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 <i>p</i> < 0.05 were considered to represent significant differences. The probability values <i>(p</i> > 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.</p